Born in the Brooklyn borough of New York City, or in Philadelphia, Pennsylvania (sources differ), the daughter of Lillian Dubois, DuBois began her acting career in theater during the early 1960s, appearing in plays such as "Golden Boy" alongside Sammy Davis Jr. and Louis Gossett Jr., and "A Raisin in the Sun". DuBois' early television acting credits include the 1969 television movie J.T. and the long–television soap opera Love of Life, on which her 1970–1972 role as Loretta Allen made her the first African–American female as a regular cast-member on a daytime serial.' In 1970, DuBois appeared in her first film, playing Vera in Diary of a Mad Housewife.

Television Producer Norman Lear saw DuBois in a Lanford Wilson play The Hot l Baltimore at the Mark Taper Forum, which led to her being cast in the 1974–1979 CBS comedy series Good Times. DuBois recorded the album Again, Ja'Net DuBois, on her Peanuts and Caviar label, in 1983. DuBois appeared in former Good Times co-star Janet Jackson's 1987 "Control" music video as her mother. In 1992, she co-starred alongside Clifton Davis in And I Still Rise, a play written and directed by Maya Angelou. She co-starred in the films I'm Gonna Git You Sucka (1988) and Charlie's Angels: Full Throttle (2003), and on television in Moesha, The Steve Harvey Show, A Different World, and The Wayans Bros..

DuBois has had at least two children: Rani Dubois, and Raj Kristo Gupta, who died of cancer in 1987 at age 26. Rani appeared on an episode of the 1970s sitcom What's Happening!!.

In 1995, DuBois won a CableAce award for Best Supporting Actress for her role in the Lifetime movie "Other Women Children". In 2000, DuBois served as Grand mMrshal for the North Amityville Community Parade and Festival Day in Amityville, New York. She won won two Emmy Awards for her voiceover work on the animated program The PJs (1999). DuBois along with the cast of Good Times received The Impact Icon Award at the 2006 TV Land Awards.