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Dubois was born in Brooklyn, New York. She has appeared in a number of other television programs (usually as a guest star), most notably in 1972, during the second season of Sanford and Son as Fred's old flame Juanita in the episode, "Sanford and Son and Sister Makes Three." Her appearance in a play of Hot l Baltimore at Mark Taper Forum got her noticed by Norman Lear, and led to her being cast in Good Times and in films.
She usually found herself playing roles which made her seem much older than her true age; when Good Times premiered in 1974, Dubois was 28 just two years older than Jimmie Walker. But her character on the show made her appear much closer in age to Esther Rolle, who was 53 at the time. According to most sources, Rolle enjoyed working with Du'Bois, despite a 25-year age difference. After John Amos' character James Evans was killed on the series, Rolle didn't like the new direction the show took, and left the show. Her departure made DuBois the de facto star of the series, alongside Walker. In 1970, Dubois played the role of a quarrelsome laundress alongside Carrie Snodgrass in the cult classic, "Diary of a Mad Housewife".
Like her late mentor Esther Rolle, Ja'net Dubois is a member of Zeta Phi Beta Sorority, Inc. In 2000, in her hometown of Amityville, New York, she was honored as the Grand Marshall for the North Amityville Community Parade and Festival Day.