Madonna, Beyoncé, Tom Cruise and Tom Hanks were among the famous faces who attended the taping of Surprise Oprah! A Farewell Spectacular at the Chicago United Centre on Tuesday.
"It's no secret that millions of people are inspired by Oprah," Madonna told the 13,000 strong crowd. "I am one of them."
Other stars in attendance included Will Smith and Jada Pinkett Smith, Katie Holmes, Rosie O'Donnell, Halle Berry, Jerry Seinfeld, Simon Cowell, Michael Jordan, Diane Sawyer and Maria Shriver.
Producers of The Oprah Winfrey Show had previously revealed that the television mogul had been kept in the dark about who would be appearing at the event, which is scheduled to air on May 23 and May 24.
"I feel the love and I thank you for it," said Winfrey, addressing the audience. "Thank you for being a part of this great night; this celebration of what you have done for The Oprah Winfrey Show.
"You have made it possible for us to stand for 25 years."
A string of performances took place across the evening from artists including Beyoncé, who performed her new single 'Run The World (Girls)', and Aretha Franklin, who took to the stage for a rendition of 'Amazing Grace'.
Other performances came from America's Got Talent runner-up Jackie Evancho, Jamie Foxx and Stevie Wonder, Josh Groban, Patti LaBelle, Rascal Flatts, Kristin Chenoweth, and Usher, who closed the show with 'Oh Happy Day'.
Additional tributes to the television mogul were paid by longterm partner Stedman Graham and her best friend Gayle King, while mentor Maya Angelou also took to the stage.
The Oprah Winfrey Show, which debuted September 8, 1986 and has run for 25 seasons, will air its final episode on May 25.
> In Pictures: Oprah Winfrey's final shows