Although Gainsbourg was born in London she was raised in Paris, France. She is the daughter of British actress and singer Jane Birkin and Jewish-French actor and singer Serge Gainsbourg. Her maternal grandmother was actress Judy Campbell and her uncle is the screenwriter Andrew Birkin, who directed her in The Cement Garden. Gainsbourg's longtime boyfriend and spouse is the French actor and director Yvan Attal, with whom she has two children, Benjamin and Alice.

Gainsbourg made her motion picture debut playing Catherine Deneuve's daughter in the film Paroles et musique (1984). She made her musical debut with her father on the song "Lemon Incest". In 1986, Gainsbourg won a César Award for "Most Promising Actress" for L'effrontée, and, in 2000, she won "Best Supporting Actress" for the film La Bûche.

In 1994, Gainsbourg made her stage debut in David Mamet's Oleanna at the Théâtre de la Gaîté-Montparnasse. Gainsbourg has sung the title song in three of her films and has released two albums.

In 1996, Gainsbourg starred as Jane Eyre in the acclaimed film adaption of Charlotte Brontë's 1847 Jane Eyre novel. The film Jane Eyre was directed by Franco Zeffirelli.

In 2004, she sang a duet with French pop star Étienne Daho on his single "If".

In 2006, Gainsbourg released her second album 5:55 to critical acclaim and commercial success, reaching the top spot on the French charts and achieving platinum status in the country.

In 2007, Gainsbourg appeared as Claire in the Bob Dylan biopic I'm Not There, also contributing a cover of the Dylan song "Just Like a Woman" to the film's soundtrack.