Jhonen Vasquez was raised in East San Jose. He attended Mount Pleasant High School, where he often spent much of his class time drawing in sketchbooks. Taking part in a contest to design a new look for his school's mascot, the Cardinal, he submitted an entry that the judges rejected. On the back of a preliminary drawing for the contest, he drew his first sketch of the character who would later become Johnny C. His high school's student newspaper published a number of his comic strips titled Johnny the Little Homicidal Maniac. Vasquez created Happy Noodle Boy while attending Mount Pleasant. According to Vasquez, "So many years ago, [my little romantical friend in high school] was the unwitting reason Happy Noodle Boy was created. [She] always asked me for comics. But I couldn't draw as fast as she requested. Thus, I tried to create the worst abomination of a comic that I could, so as to make her not want comics anymore. That abomination, my friends, was Happy Noodle Boy".

While Vasquez read his older brother's superhero comics as a child, he first became interested in the medium through the original independent Teenage Mutant Ninja Turtles comics by Peter Laird and Kevin Eastman.

After graduating in 1992, Vasquez went on to become a film student at De Anza College in Cupertino, California. Though he had little formal artistic training, he soon dropped out of De Anza to pursue a career as a professional cartoonist. He met Roman Dirge, Rosearik Rikki Simons, and Tavisha Wolfgarth-Simons at Alternative Press Expo in 1995. Dirge later became a writer on Vasquez's Invader Zim, while Rikki Simons became the voice of the show's crazed robot GIR, as well as a member of the show's coloring team. Rikki Simons also worked with Vasquez on the coloring seen in his two-issue comic "I Feel Sick".

By September 1996, Vasquez announced in his introductory text to the sixth issue of Johnny the Homicidal Maniac, he had reached sufficient success in his artistic career to be able to quit his day-job and devote himself full-time to his art.

Many of the characters in Vasquez's cartoons are highly geometric and thin nearly to the point of being stick figures. The protagonists in his comics are typically insane characters who live in dysfunctional societies, and whose manias are able to speak through other objects (as with Johnny and the Doughboys, or Devi and Sickness.) His storylines tend to follow the basic black comedy formula. Smiley faces are often found in his artwork, trying to evoke an ironic sense of happiness in a world of chaos and darkness.

His comic works often feature an outside narrative in the form of notes and comments left in the corners of his strips. This can be found in the vast majority of Vasquez's comics, such as in issue #5 of JTHM: A large monster is shown bursting through a wall, arms and hands flailing, tentacles sweeping through the air. It is a scene that surely conveys a sense of violence and danger, yet in the corner of the panel, a small box contains the text "Kids - Don't be scared! He don't bite!" These small touches help with emotional connection to Vasquez's work, and are likely one of the factors in his cult following.

Carpe Noctem magazine published early one-page strips featuring Johnny in the early 1990s. In 1995, Slave Labor Graphics began publishing a series of Johnny comics after Vasquez submitted samples of his artwork to them. Vasquez's first comic, Johnny the Homicidal Maniac, ran for seven issues and was collected as a hardcover and a trade paperback book, Johnny the Homicidal Maniac: Director's Cut. The cover features the logo "Z?", meaning "question sleep", which appears frequently throughout Vasquez's work and relates to his characters' insomnia and his own hypnophobia. The series follows Johnny as he searches for meaning in his life, a quest that frequently leads to the violent deaths of those around him as well as, briefly, his own. A photograph of one of Vasquez's friends, Leah England, serves as the middle of a portrait collection on the cover for the second issue of Johnny the Homicidal Maniac. England also gave Vasquez the inspiration for a filler strip about a child who was dangerously afraid of losing sight of his mother, as well as the notorious "Meanwhile" filler piece in the second issue of JTHM.

Vasquez's next project was The Bad Art Collection, a 16-page one-shot comic. Vasquez stated that he did the book's art while he was in high school to discourage classmates from asking him to draw for them.

In 1997, Vasquez gave Squee, a supporting character from JTHM, his own four-issue series. It chronicles Squee's encounters with aliens, Satan's son, and eventually Satan himself. The trade version (which features a cover image of Squee with the words "Buy me or I'll die!") contains, in addition to the actual Squee comics, the Meanwhiles that were left out of the Director's Cut of JTHM, as well as comics of Vasquez's "real life" and Wobbly-Headed Bob.

Vasquez's next project was I Feel Sick, colored by Rikki Simons. I Feel Sick follows a tortured artist named Devi (another character introduced in JTHM) as she tries to maintain her sanity in an insane vision of society, despite conversing with Sickness, one of her own paintings.

Slave Labor has published three Fillerbunny mini comics, the third having been released in March 2005. The mini comic was a spin-off of a filler comic designed to replace a vacant page usually reserved for advertising space in the Squee! comics. Vasquez said at the 2007 New York ComicCon that the original Fillerbunny comics would be done in a single night and he would rush through and do whatever he could in a small amount of time. The third issue, however, broke this mold. According to the introduction, it took over nine months to complete, and he feels it is of much higher quality than the first two.

At Comic-Con 2005, Vasquez mentioned that his next comic was a love story. Since this, however, he attended an event in early 2007 and stated he was not working on his 'own' comics - he was collaborating on two comics in the style of Everything Can Be Beaten, acting only as author. The first, titled Jellyfist was intended for release on July 25, 2007. However, the initial print run of Jellyfist was incredibly poor, and so it was re-released in October 2007.

After the success of Squee!, the children's cable network Nickelodeon approached Vasquez about producing an animated television series. The series, Invader Zim, was canceled after little more than a year; only 27 half-hour episodes were made, most split into two 11-minute episodes but several full half-hours, and many episodes unfinished. Episodes in the second season aired first internationally and later aired on Nicktoons Network in 2006. The show was cancelled despite its good ratings, Invader ZIM - TV.com ostensibly due to its dark tone and violence, although in the DVD commentary Vasquez said he would never go back to Viacom. He later admitted at a 2005 San Diego Comic-Con that he would love to continue animation again as long as it's with a network and people that he trusts. They had signed a contract agreeing that they would make a series finale (which was intentionally the unfinished movie, "Invader Dib"). Vasquez states in the commentary that September 1 is Zim Day, which is also his birthday.

AnimeWorks, a branch of Media Blasters, released the DVD collection Invader Zim Vol. 1 on May 11, 2004. It contains the first nine episodes plus audio commentary by Vasquez and various cast- and crew-members, including Richard Steven Horvitz, Rosearik Rikki Simons, Melissa Fahn, Wally Wingert, Andy Berman, and Kevin Manthei. The company released Vol. 2 on August 31, 2004, Vol. 3 on October 12, 2004, and a boxed set was released on April 12, 2005. The boxed set contained a "Special Features" DVD with audio-only episodes never aired on Nickelodeon, as well as the original uncut version of the Christmas special.

Although Invader Zim merchandise has been an immensely successful franchise at Hot Topic since the show was on air, Vasquez makes no profit from sales of such. All rights to Invader Zim merchandise are owned solely by the Nickelodeon television network, which does not consult Vasquez regarding the designs of said products.

Invader Zim has also run on the cable channels Nicktoons Network, YTV (a Canadian youth network) and MTV2 (in the "'Sic'Emation" block of the latter) and is available on iTunes in the United States and in Canada as well as Netflix.

Vasquez also directed the music video for "Shut Me Up" by the band Mindless Self Indulgence which centers around a store clerk having a "meltdown." In the music video, you can see Vasquez's characters, Happy Noodle Boy, on top of the aisles and Fillerbunny behind the counter.

Vasquez collaborated with Crab Scrambly to produce the storybook Everything Can Be Beaten, published by Slave Labor in 2002. Vasquez, credited as Chancre Scolex, wrote the story and Crab Scrambly illustrated it. Everything Can Be Beaten is about a strange person who lives in a room in which he can do nothing but beat kittens. However, an adventure into the outside world changes his perspective, and he discovers that "everything can be beaten." The storybook inspired Urban Squall in 2008 to create the puzzle browser game Bloody Fun Day.

Vasquez did the entire artwork for the deluxe edition of the new Mindless Self Indulgence album "If" as well as the digital single, "Mastermind".

He has also indicated that he shows his artwork in galleries from time to time.

He also did an art rendition of Bioshock 2 called "The Sisters". BioShock 2's He was quoted saying the following about the rendition:

Vasquez's writing often conveys misanthropic and pessimistic themes, often used for the purposes of parody and satire. Similar styles and mannerisms can be found in many of his characters as well as running gags and common themes, including repeated references to moose, meat, bees, chihuahuas, monkeys, tacos, pigs, cheese, morbid obesity, and "dookie". Vasquez also frequently sneaks cameo appearances of characters such as Happy Noodle Boy and Johnny The Homicidal Maniac as well as himself, into unrelated works. His influences include David Cronenberg, Chester Brown, Kurt Vonnegut, Franz Kafka, H. R. Giger, Terry Gilliam, and David Lynch.

Several of Vasquez's works have featured gothic characters or depictions of the goth subculture for the purpose of satire. In an interview on the show The Screen Savers, Vasquez responded to host Kevin Pereira's comment that fans considered him "a goth king," saying disdainfully: "King, yeah, but goth... I mean, that's just arrogant."