In 2008, Eisenberg was honored alongside Olivia Wilde with the Vail Film Festival Rising Star Award. In 2009, he starred in the comedy dramaAdventureland and the horror comedy Zombieland, for which he won critical acclaim. He then played Facebook co-founder Mark Zuckerberg in The Social Network (2010), for which he received a Golden Globe and Academy Award nomination in the Best Actor category. Since then, he voiced the lead role inRio (2011), and starred in the adventure comedy 30 Minutes or Less (2011). 

Eisenberg was born in Queens, New York City, the son of Amy, who worked as a clown at children's parties, and Barry Eisenberg, who ran a hospital and later became a college professor. He has two siblings, Hallie Kate Eisenberg, a former child actress who was once famous as the "Pepsi girl" in a series of commercials, and Kerri. He was raised in a secular Jewish family that originated in Poland and Ukraine.[6][7] Eisenberg grew up in Queens andEast Brunswick Township, New Jersey, attending the East Brunswick Public Schools at Frost School, Hammarskjold Middle School, Churchill Junior High School, and spending his sophomore year at East Brunswick High School.

Eisenberg struggled to fit in at school and began acting in plays at the age of 10, stating "when playing a role, I would feel more comfortable, as you're given a prescribed way of behaving."[9] Eisenberg studied contemporary architecture at The New School in New York City's Greenwich Village neighborhood. Originally, he had applied and was accepted to New York University, but declined enrollment in order to complete a film role.

Eisenberg's first professional role was in Arje Shaw's off-Broadway play, The Gathering. Eisenberg made his Broadway debut as an understudy in a 1996 revival of Summer and Smoke. He made his screen debut on the 1999 television series Get Real, which was canceled in 2000. In 2001, Eisenberg appeared in a Dr. Pepper commercial in the UK. After appearing in the made-for-television filmLightning: Fire from the Sky, he starred in the independent film Roger Dodger (for which he won an award at the San Diego film festival), and in The Emperor's Club, both of which were released in 2002 to generally positive reviews.

In 2005, Eisenberg appeared in Cursed, a horror film directed by Wes Craven, and The Squid and the Whale, a well-reviewed independent drama also starring Laura Linney and Jeff Daniels. In 2007, he starred opposite Richard Gere and Terrence Howard in The Hunting Party, a comic thriller in which he plays a young journalist in Bosnia. In 2009, Eisenberg played the lead role in Adventureland, a comedy directed by Greg Mottola and filmed in Kennywood Park near Pittsburgh, Pennsylvania. Filming ended in October 2007,[12] and the movie premiered at the Sundance Film Festival in 2009. In November 2007, Eisenberg was cast in the indie comic-drama Holy Rollers. He played a young Hasidic Jew who gets lured into becoming an ecstasy dealer. Filming took place in New York in 2008. During the late 2000s, he also had roles in the independent films Solitary Man, playing Cheston, and Camp Hope, a horror film directed by George Van Buskirk.

Eisenberg's breakthrough starring role was as the neurotic Columbus in Zombieland. The horror comedy, which paired him with Woody Harrelson on a roadtrip through a post-zombie apocalypseAmerica, was a surprise hit. In 2010, he starred alongside Andrew Garfield in the role of Facebook creator Mark Zuckerberg in the film The Social Network, for which he earned the Best Actor Awardfrom the National Board of Review of Motion Pictures, and nominations for Best Actor at the Golden Globes and Academy Awards.

On November 22, 2010, Eisenberg was honored, along with Whoopi Goldberg, Joycelyn Engle and Harvey Krueger, at the Children at Heart Celebrity Dinner Gala and Fantasy Auction, to benefit The Children of Chernobyl. Steven Spielberg is Chair of the event each year. On January 29, 2011, Eisenberg hosted Saturday Night Live on NBC, with musical guest Nicki Minaj. During his opening monologue, Eisenberg was joined by Facebook creator Mark Zuckerberg.

In 2011, he starred in the box-office animated hit Rio, as the main character Blu, a Spix's Macaw who learns how to fly, starring alongside Anne Hathaway, his former co-star (and onscreen sibling) fromGet Real, George Lopez, Jake T. Austin, Tracy Morgan, Jemaine Clement, Leslie Mann, Rodrigo Santoro, will.i.am, and Jamie Foxx. He contributed his singing voice for one of the songs in the soundtrack, "Real in Rio". He also starred in 30 Minutes or Less, released August 2011.

Eisenberg is set to make his playwriting debut Off-Broadway in Rattlestick Playwrights Theater's production of Asuncion, presented at Cherry Lane Theatre. Eisenberg will act in the play that will be directed by Kip Fagan. The play highlights two liberal-minded friends whose assumptions are challenged by their new Filipina roommate, played by Camille Mana.

In 2012, he will star alongside Melissa Leo in Predisposed, a drama about a drug addict mother and her piano prodigy son[19] and in the romantic comedy The Bop Decameron.

Eisenberg plans to write for stage and screen.

Eisenberg has said that fan confusion between himself and Michael Cera is annoying.

Eisenberg is fond of cats and has been involved in fostering the animals. He has been associated with Farm Sanctuary and has presented as several of their galas. Jesse has mentioned that he has obsessive–compulsive disorder.