Berg was born Tilly Edelsteinin Harlem, New York City, and attended public schools. She married Lewis Berg in 1918; they had two children, Cherney (1922—2003) and Harriet (1926—2003). She learned theater while producing skits at her father's Catskills Mountains resort in Fleischmanns, New York. She developed a semi-autobiographical skit, portraying a Jewish family in the New York tenements, into a radio show. On November 20, 1929, a 15-minute episode of The Rise of the Goldbergs was first broadcast on the NBC radio network. She started at $75 a week. Less than two years later, in the heart of the Great Depression, she let the sponsor propose a salary and was told, "Mrs. Berg, we can't pay a cent over $2,000 a week."

Berg became inextricably identified as Molly Goldberg, the bighearted matriarch of her fictitious New York family who moved to Connecticut as a symbol of Jewish-American upward mobility. She wrote practically all the show's radio episodes (more than 5000) plus a Broadway adaptation, Me and Molly (1948). It took considerable convincing, but Berg finally prevailed upon CBS to let her bring The Goldbergs to television in 1949. Berg won the first ever Emmy Award for Lead Actress in a Comedy Series her debut year on the network...her twentieth consecutive year of playing the role...and the show stayed in production for five years.

The Goldbergs ran into trouble in 1951, during the McCarthy Era. Co-star Philip Loeb (Molly's husband, patriarch Jake Goldberg) was one of the performers named in Red Channels: The Report of Communist Influence in Radio and Television and blacklisted as a result. Loeb resigned rather than cause Berg trouble. Reportedly he received a generous severance from the show, but it wasn't enough to prevent him from sinking into the depression that ultimately drove him to suicide in 1955.The Complete Directory to Prime Time Network and Cable TV Shows (6th Ed.), by Tim Brooks and Earle Marsh, p. 404 The Goldbergs returned a year after Loeb departed the show and continued until 1954, after which Berg also wrote and produced a syndicated film version. The show remained in syndicated reruns for another few years, after one year of production and 39 episodes (it aired on some stations as Molly).

In 1959, Berg won the Tony Award for Best Actress for her performance in A Majority of One. In 1961 she won the Sarah Siddons Award for her work in Chicago theatre. Berg also published a best-selling memoir, Molly and Me, in 1961. That same year, she made one last stab at television success in the Four Star Television situation comedy Mrs. G. Goes to College (retitled The Gertrude Berg Show at midseason). Her costars were Cedric Hardwicke, Mary Wickes, and Marion Ross. Berg played a 62-year-old widow who decides to attend college.

Berg was also a songwriter. Her composition "That Wonderful Someone" even found its way into the repertoire of country music singer Patsy Cline, appearing on her 1957 debut album.

A biography of Berg, Something on My Own: Gertrude Berg and American Broadcasting, 1929—1956, by Glenn D. Smith, Jr. (Syracuse University Press) appeared in 2007. Aviva Kempner's 2009 documentary, Yoo-Hoo, Mrs. Goldberg, deals with Berg's career, and to an extent, her personal life.

Berg died of heart failure on September 14, 1966 in a Manhattan hospital. She is buried in Clovesville Cemetery. Her husband, Lewis, died in 1985, aged 87.