In September 2007, Cuoco began starring in the CBS sitcom The Big Bang Theory, playing Penny, a Cheesecake Factory employee and aspiring actress who lives across the hall from physicists Dr. Leonard Hofstadter and Dr. Sheldon Cooper. Prior to the 2010–2011 season, she earned US$60,000 an episode for the series, the same as her co-stars. In 2010, the cast negotiated a raise for each actor to make US$200,000 per episode. As of August 2014, Cuoco and her co-stars from The Big Bang Theory Johnny Galecki and Jim Parsons each earned an estimated US$1 million per episode.
Cuoco starred in the Lifetime film To Be Fat like Me, which premiered on January 8, 2007. She played a minor role in the Prison Break episodes "The Message" and "Chicago", as well as starring in the comedy horror spoof Killer Movie in 2008. She also appeared in the 2010 film The Penthouse. In 2011, she appeared in Allure magazine's annual "Naked Truth" feature. That same year, she appeared in the films Hop and The Last Ride. She was also chosen to host the Teen Choice Awards in 2011, as well as the People's Choice Awards in 2012 and 2013. She is a regular on Priceline.com commercials as the daughter of William Shatner's character.
In 2007, while working on The Big Bang Theory, Cuoco privately dated co-star Johnny Galecki for roughly two years, until December 2009. She later told CBS Watch in September 2010 that they have remained on good terms since ending their relationship.