Holmes was born in February 1984 in Washington, D.C. She is the daughter of government Service worker Christian Holmes IV and congressional committee staffer Noel Daoust. When she was 9 years old, Holmes and her younger brother Christian Holmes V moved to Houston, Texas due to her family's job relocation. She wrote a letter to her Father about the move saying, "What I really want out of life is to discover something new, something that mankind didn't know was possible to do." Holmes studied Mandarin as a child and completed three years of summer language classes at Stanford University before graduating from high school.

She attended St. John's School in Houston and was recognized for her "tireless optimism and a particularly warm smile." During high school, Holmes was interested in computer programming and started her first Business selling C++ compilers to Chinese universities. In 2001, Holmes applied to Stanford University and was named a President's Scholar, which came with a stipend to use on a research project. She studied chemical engineering and used the stipend to work in a lab with Ph.D. candidates and Channing Robertson, dean at the engineering school.

After the end of her freshman year, Holmes worked in a lab at the Genome Institute of Singapore on testing for severe acute respiratory syndrome (SARS) through the collection of blood samples with syringes. She filed her first patent on a wearable drug-delivery patch in 2003. In March 2004, she dropped out of Stanford's School of Engineering and used her tuition money as seed funding for a consumer Health care Technology company.

By December 2004, she had raised $6 million to fund Theranos. The company's first revenue came from contracts Holmes established with pharmaceutical companies to conduct testing and other clinical trials. By the end of 2010, Holmes had more than $92 million in venture capital for Theranos. In July 2011, Holmes was introduced to former Secretary of State George Shultz. After a two-hour meeting, he joined the Theranos board of Directors. She was recognized for forming "the most illustrious board in U.S. corporate history" over the next three years. Holmes operated Theranos in stealth mode without press releases or a company website until September 2013 when the company announced a partnership with Walgreens to make in-store blood sample collection centers.

Media attention increased in 2014 as she was on the cover of Fortune, Forbes, T: The New York Times Style Magazine and Inc., who considered her "The Next Steve Jobs". Forbes recognized Holmes as the world's youngest self-made female Billionaire and ranked her #110 on the Forbes 400 in 2014. Theranos was valued at $9 billion with more than $400 million in venture capital. By the end of 2014, she had 18 U.S. patents and 66 foreign patents in her name.

In 2015 She was awarded the Honorary degree of Doctor of Humane Letters (DHL) from Pepperdine University in Malibu, California, She also served as the Commencement speaker at that ceremony. As of March 2018 her honorary degree has not been revoked.

Following the scrutiny over the Theranos lab controversy, Fortune named Holmes one of the "World's Most Disappointing Leaders" for 2016.

On May 16, 2017, approximately 99% of Theranos shareholders reached an agreement with the company to dismiss all current and potential litigation in exchange for shares of preferred stock. Holmes released a portion of her equity to offset any dilution of stock value to non-participating shareholders.

Prior to the March 2018 settlement, Holmes held a 50-percent stock ownership in Theranos. Forbes listed her as one of "America's richest Self-Made Women" in 2015 with a net worth of $4.5 billion. In June 2016, Forbes released an updated valuation of $800 million for Theranos, which made Holmes’s stake essentially worthless, because other Investors owned preferred shares and would have been paid before Holmes, who owned only Common stock. Holmes reportedly owes a $25 million debt to Theranos in connection with exercising options. She did not receive any company cash from the arrangement, nor did she sell any of her shares, including those associated with the debt.