Marquis grew up in Walnut, Illinois. His brother David died in 1892 at the age of 20; his father James died in 1897. After graduating from Walnut High School in 1894, he attended Knox Academy, a now-defunct preparatory program run by Knox College, in 1896, but left after three months. From 1902 to 1907 he served on the editorial board of the Atlanta Journal where he wrote many editorials during the heated election between his publisher Hoke Smith and future Pulitzer Prize winner, Clark Howell (Smith was the victor).

During 1909, Marquis married Reina Melcher, with whom he had a son, Robert (1915-1921) and a daughter, Barbara (1918-1931). Reina died on December 2, 1923. Three years later he married the actress Marjorie Potts Vonnegut. She died in her sleep on October 25, 1936. Marquis died of a stroke after having suffered three earlier strokes that partly disabled him.

On August 23, 1943, the United States Navy christened a Liberty ship, the USS Don Marquis , in his memory.

Marquis began work for the newspaper The Evening Sun during 1912 and edited for the next eleven years a daily column "The Sun Dial.". During 1922 he left The Evening Sun (shortened to The Sun in 1920) for the New York Tribune (renamed the New York Herald Tribune in 1924), where his daily column, "The Tower" (later "The Lantern") was a great success. He regularly contributed columns and short stories to the Saturday Evening Post, Collier's and American magazines and also appeared in Harper's, Scribners, Golden Book, and Cosmopolitan.

Marquis's best-known creation was Archy, a fictional cockroach (developed as a character during 1916) who had been a free-verse poet in a previous life, and who supposedly left poems on Marquis's typewriter by jumping on the keys. Archy usually typed only lower-case letters, without punctuation, because he could not operate the shift key. His supposed writings were a type of social satire, and were used by Marquis as a newspaper column named "archy and mehitabel" (Archy's best friend was a fictional alley cat named Mehitabel). Other characters developed by Marquis included Pete the Pup, Clarence the ghost, and an egomaniacal toad named Warty Bliggins.

Marquis was the author of about 35 books. He co-wrote (or contributed posthumously) to the films The Sports Pages, Shinbone Alley, The Good Old Soak and Skippy. The 1926 film The Cruise of the Jasper B was supposedly based on his 1916 novel of the same name, although the plots have little in common.