Longoria was born in Corpus Christi, Texas, on March 15, 1975, the youngest of the four daughters of her Tejano parents Ella Eva (Mireles) and Enrique Longoria Jr. One of her sisters had special needs. Longoria told Redbook in 2016:

There was never any resentment, because I saw how hard my mother worked and all she did for my sister, and I wanted to do whatever I could to help. We all did.

She was raised Roman Catholic. She did not speak Spanish growing up, and did not learn the language until 2009. While in high school, she worked at a Wendy's restaurant part-time for three years to help earn money for her quinceañera. Longoria previously told the press how her years in fast food as a teen shaped her work ethic, saying "I couldn't wait to get to work and make my own money." Longoria earned her Bachelor of Science degree in kinesiology at Texas A&M University-Kingsville. At that time (1998), she also won the title of Miss Corpus Christi USA. After completing college, she entered a talent contest that led her to Los Angeles; and shortly thereafter, was spotted and signed by a theatrical agent. While auditioning for roles, she worked as a headhunter for four years. Longoria has publicly credited Jennifer Lopez as being an inspiration to her early in her career. “Jennifer broke down so many doors so that we could walk through them,” she told the magazine PEOPLE. “And she kicked them down. They were not easy doors.”  She also described Tejana legend Selena as being "my idol, my inspiration" and "the reason I even dared to dream that a better life was possible." After three years at California State University, Northridge, she received her master's degree in Chicano studies in May 2013. Her thesis was "Success STEMS From Diversity: The Value of Latinas in STEM Careers".

Longoria guest-starred in an episode of Beverly Hills, 90210. Another guest appearance on General Hospital the same year led to a contract role on the CBS Daytime soap opera The Young and the Restless, where she portrayed Isabella Braña from 2001 to 2003. After leaving that show, she appeared in the 2003 revival of Jack Webb's long-running Dragnet media franchise. She then starred in Señorita Justice, a poorly received direct-to-video film; and a television film, The Dead Will Tell.

In 2003, Longoria was cast as Gabrielle Solis in the ABC comedy-drama Desperate Housewives. She later commented, "I think it's funny when people say I'm an overnight sensation, because I've been working at it for 10 years." Shortly after her debut on Desperate Housewives, she starred in a direct-to-video film, Carlita's Secret, for which she was also co-producer.

In 2006, she was nominated for a Golden Globe Award for Best Performance by an Actress in a Television Series – Musical or Comedy, along with her co-stars in Desperate Housewives. That year, she received the ALMA Award, and was named entertainer of the year. She also starred opposite Michael Douglas and Kiefer Sutherland in the thriller The Sentinel (2006)—her first major role in a theatrical feature film—and played Sylvia in Harsh Times, starring Freddy Rodríguez and Christian Bale.

In March 2008, Longoria opened the restaurant Beso (which means "kiss" in Spanish) in Hollywood, along with partner and celebrity chef Todd English. The restaurant is located on Hollywood Boulevard and is accompanied by Kiss, a Victorian-themed nightclub on the floor above. The Hollywood Beso was scheduled to be the focus of a pilot episode for a tentative reality series called Beso: Waiting on Fame, to air on VH1 in late 2010. In 2009, Longoria and various investor-partners opened a Beso restaurant, with a nightclub called Eve above it, in the Crystals retail and entertainment district of CityCenter in Las Vegas.