Gilbert decided at a young age that she wanted to be an actress after seeing her older sister Melissa Gilbert star in Little House on the Prairie. The roles she has played tend to reflect her laid back and unthreatening personality. Following appearances in television movies and a commercial for Kool Aid, at the age of thirteen she landed the role of Darlene Conner, the sarcastic middle child, in Roseanne. Sara was a constant cast member during the show's nine-year run from 1988 to 1997, for which she also wrote an episode called Don't Make Me Over. Sara's contribution was considered so important to Roseanne that the show's producers arranged the filming schedule to allow her to study at Yale University while remaining part of the cast. At university, she majored in art with an emphasis on photography and she graduated with honors in 1997. To coincide with her personal life, Sara wanted her Roseanne character Darlene to come out as a lesbian, although ABC executives were not ready for a lesbian in a prominent role. Gilbert has also made guest appearances in many TV shows such as The Simpsons, 24, ER, and Will & Grace . She returned to prime time television in the fall of 2005 in the sitcom Twins on The WB network.

Gilbert has appeared in two films alongside Drew Barrymore, in Poison Ivy in 1992 and Riding In Cars With Boys in 2001. She has had several other minor roles following Roseanne, including short film $30 (aka 30 Bucks) as part of the Boys Life 3 feature. She directed her own short film Persona Non Grata in 1998. She also made a cameo appearance in the film High Fidelity in 2000, appearing alongside Jack Black and John Cusack.