'I will not be taking a paternity test, ever,' Oprah told a reporter outside Four Seasons hotel in Manhattan.
An 84-year-old Navy veteran named North Robinson had claimed to the New York Post that he was Oprah's biological father, reports News.com.au.
Robinson hails from Kosciusko, Missouri, a small rural town that Oprah left when she was six.
'I've never heard of him. I know who is claiming to be my real father,' Oprah said.
Robinson said that he did not want any share in Oprah's riches but just wants to meet his so-called daughter before he dies.
When Winfrey was asked what Robinson would get out of calling her his daughter, 'Get out of my face', replied Oprah before getting into an SUV.
Oprah considers Vernon Winfrey as her father. Vernon is Oprah's mother Vernita Lee's longtime boyfriend.
But the 56-year-old talk show queen has also insisted that Vernon is not her biological father.
Photograph: Lucas Jackson/Reuters