The talk show host and actress sparked speculation that she could be in line for the job following her rousing speech at the Golden Globes earlier in January, with many high profile stars backing her for the post.
But Winfrey (63) has now said she has no desire to put herself forward as a Democratic candidate for the 2020 election.
She told US magazine InStyle: "I've always felt very secure and confident with myself in knowing what I could do and what I could not.
"And so it's not something that interests me. I don't have the DNA for it."
Referring to her best friend, CBS News anchor for This Morning Gayle King, Winfrey said: "Gayle - who knows me as well as I know myself practically - has been calling me regularly and texting me things, like a woman in the airport saying, 'When's Oprah going to run?'.
"So Gayle sends me these things, and then she'll go, 'I know, I know, I know! It wouldn't be good for you - it would be good for everyone else'."
She added: "I met with someone the other day who said that they would help me with a campaign. That's not for me."
Winfrey made an impassioned call for "a brighter morning even in our darkest nights" at the Golden Globes, prompting her peers and fans to call for her to run for office.
As she became the first black woman to win the Cecil B. DeMille Award, Winfrey told the audience: "For too long, women have not been heard or believed if they dared to speak their truth to the power of those men, but their time is up."
She added: "I want tonight to express gratitude to all the women who have endured years of abuse and assault because they, like my mother, had children to feed and bills to pay and dreams to pursue."
Meryl Streep and Tom Hanks were among the stars who shared their backing for Winfrey.
Donald Trump said earlier this month that he would "beat Oprah" if she ran against him at the next election.