She moved to New York City after graduating from MICA in 2006; she began taking classes with the Atlantic Theater Company and the Upright Citizens Brigade Theatre, where she met Ilana Glazer. Jacobson is bisexual.
From 2009 to 2011, Jacobson and Glazer wrote and performed in a web series titled Broad City, which focused on their lives in New York. The series was nominated for an ECNY Award for Best Web Series. It was well received by critics and developed a cult following. At the Upright Citizens Brigade, Jacobson and Glazer adapted the series into a live show that they performed in, called Broad City Live.
In 2011, Jacobson wrote and performed in a solo show called Welcome to Camp, which ran in New York and Los Angeles.
In 2013, Jacobson published two coloring books with Chronicle Books: Color This Book: New York City and Color This Book: San Francisco. Jacobson also illustrated a book titled Carry This Book, published October 2016 by Viking Press. It features colorful, humorous illustrations of the imagined contents of various celebrities' bags. “I have always been intrigued by what people carry around with them. It can tell you everything,” says Jacobson in the book's introduction. Well-received by critics, Carry This Book was a New York Times bestseller.
In February 2014, Comedy Central renewed the show for a second season. Season two received positive reviews, with Metacritic giving it a score of 89 out of 100, based on reviews from 8 critics, indicating "universal acclaim." Rotten Tomatoes gave the second season a rating of 100%, based on reviews from 11 critics, with the site's consensus: "Led by two of the funniest women on TV, Broad City uses its stars' vibrant chemistry to lend an element of authenticity to the show's chaotic yet enlightening brand of comedy."
In January 2015, the series was then renewed for a third season, which premiered on February 17, 2016. In January 2016, the series was renewed for a fourth and a fifth season.
In 2017, Jacobson hosted a 10-episode podcast about modern and contemporary art called A Piece of Work co-produced by The Museum of Modern Art and WNYC Studios.
The show has received critical acclaim from fans and critics alike. Review aggregation website Metacritic noted season 1 received "generally favorable reviews," giving it a score of 75 out of 100, based on reviews from 14 critics. Karen Valby from Entertainment Weekly described the show as a "deeply weird, weirdly sweet, and completely hilarious comedy." The Wall Street Journal referred to the show as "Sneak Attack Feminism." Critic Megan Angelo quotes Abbi Jacobson, main star of Comedy Central's Broad City: "If you watch one of our episodes, there’s not a big message, but if you watch all of them, I think, they’re empowering to women.” The A.V. Club critic Caroline Framke wrote that Broad City was "worth watching" despite its "well-trod premise," and that the series is "remarkably self-possessed, even in its first episode."