Tautou was born in the Puy-de-Dôme département of Auvergne, and was raised in Montluçon in nearby Allier, Auvergne. Her father is a dental surgeon and her mother is a teacher. Tautou showed an interest in comedy at an early age and started her acting lessons at the Cours Florent. She is one of several famous actors to have attended Cours Florent (others including Isabelle Adjani, Daniel Auteuil and Guillaume Canet). After graduating she went on to star in some of French cinema's biggest and most famous films.

In 1998, Tautou participated in a Star Search-like competition sponsored by Canal+ called "Jeunes Premiers" (The Young Debut) and won Best Young Actress at the 9th Béziers Festival of Young Actors. Then, she came to the attention of Tonie Marshall, who gave her a role in the César-winning Venus Beauty Institute (1999, aka Vénus beauté (institut)). In 2000, she won the Prix Suzanne Bianchetti as her country's most promising young film actress. In 2001 Tautou rose to international fame for her performance as the eccentric lead in the romantic comedy Le Fabuleux Destin d'Amélie Poulain (Amélie). In June 2004, she was invited to join the Academy of Motion Picture Arts and Sciences (AMPAS).

In 2005, Tautou worked in her first full Hollywood production, opposite Tom Hanks, in the film version of Dan Brown's best-selling novel The Da Vinci Code, directed by Ron Howard and released in May 2006. She acted alongside Gad Elmaleh in Pierre Salvadori's Hors de prix (Priceless), released 13 December 2006. The film has been compared to Breakfast at Tiffany's.

Tautou starred with Guillaume Canet in Claude Berri's Ensemble, c'est tout in 2007, an adaptation of the eponymous novel by Anna Gavalda.

Tautou played the lead role in the biopic of fashion designer Coco Chanel. Filming began in Paris in September 2008, and released in France on April 22, 2009. The film is titled Coco avant Chanel, and is directed by Anne Fontaine. The script of the movie is partially based on Edmonde Charles-Roux’s book “L’Irrégulière” (”The Non-Conformist”). As part of promoting the film, Tautou was named as the next spokesmodel for Chanel No. 5, replacing Nicole Kidman. She was directed in the advertisement by Jean-Pierre Jeunet, with whom she worked on Amélie and A Very Long Engagement. The advertisement was released in 2009 to coincide with the film's release.

She appeared in the video of "I Love Your Smile", a song by British singer-songwriter Charlie Winston.

Her favourite authors are Victor Hugo, Oscar Wilde, Paul Auster, and Timothy Zahn; and her favourite poets are Charles Baudelaire and Tristan Tzara. She was brought up attending church, though she has now stated that she is "not officially" a Catholic. She is fascinated by monkeys. She takes pictures of each reporter who interviews her and keeps them in a scrapbook. Tautou says she still considers France her base, and plans to pursue a career predominantly there rather than crossing over to the United States. As she told Stevie Wong of The Straits Times:

After the premiere of the film Amélie (for which she received phenomenal amounts of paparazzi and press coverage) she travelled to the jungles of Indonesia to help with the preservation of a monkey sanctuary.

The song "Tautou" by Brand New, from the album Déjà Entendu ("previously heard") is named after her.