Lee Bo-young began her career in the industry with winning the Daejeon entry for the Miss Korea beauty pageant in 2000. Having majored in Korean literature at Seoul Women's University, she originally dreamed of becoming a news presenter. She was one of 15 finalists in MBC's annual recruitment drive in 2002, but ended up not getting the job. Instead, she turned to modeling, and was chosen as the exclusive advertising model for Asiana Airlines, considered a gateway to stardom.

Lee made her acting debut in 2003, and among her early roles were in People of the Water Flower Village (2004) opposite Song Il-gook, and as the antagonist in Save the Last Dance for Me (2004).

After playing the love interest in My Brother (2004) and A Dirty Carnival (2006), Lee was cast in her first major big screen role in Once Upon a Time (2008). The heist comedy is set during colonial rule in the 1940s, and she and Park Yong-woo played notorious swindlers out to steal a Diamond from the Japanese army.

In 2005, Lee played her first leading role in the daily drama My Sweetheart, My Darling, followed by the Silla-set historical epic Ballad of Seodong with Jo Hyun-jae. She spent the next several years in television dramas — Mr. Goodbye with Ahn Jae-wook, Queen of the Game with Joo Jin-mo, and Becoming a Billionaire with Ji Hyun-woo — but none struck a chord with audiences or critics.

Her two films released in 2009 were both melodramas. In More Than Blue, Lee plays the soulmate of a terminally ill man (Kwon Sang-woo), but their love ends in a tragedy undercut with a poetic sparseness. While in the ironically titled I Am Happy, Lee played a nurse who falls for a patient in the psychiatric ward (Hyun Bin), and in each other they find consolation to see them through their harsh and miserable reality.

Lee returned to television in 2010, playing a seductress in the quirky cable drama Harvest Villa opposite Shin Ha-kyun. She then had a guest arc as the president's daughter in spy series Athena: Goddess of War, followed by post-divorce romance series Hooray for Love with Lee Tae-sung, and revenge drama Man from the Equator (2012) with Uhm Tae-woong.

Lee met actor Ji Sung on the set of 2004 TV series Save Last Dance for Me, and they confirmed their relationship in 2007. On August 2, 2013, they announced their engagement by uploading handwritten letters on their respective official fan sites. They married at Aston House, W Seoul Walkerhill Hotel on September 27, 2013. Their first child, daughter Kwak Ji-yoo was born on June 12, 2015.

In 2014, Lee played a mother who travels back in time two weeks before her daughter's kidnapping and murder in God's Gift - 14 Days.

While on acting hiatus in 2015 during her pregnancy, she published the book Time of Love, a collection of essays on 23 books with themes of love, consolation and growth that have impacted Lee throughout her childhood and adulthood; they include The Little Prince, José Mauro de Vasconcelos's My Sweet Orange Tree, and Alain de Botton's Essays in Love.

Lee was confirmed to host 2017 SBS Drama Awards together with Shin Dong-yup. Previously, Lee has emceed the same award ceremony in 2013 together with Lee Hwi-jae and Kim Woo-bin.

In 2018, Lee starred in the Korean adaption of the Japanese drama Mother, playing the titular character.