Winfrey, 69, the host of the long-running The Oprah Winfrey Show, as well as a businesswoman and philanthropist, said on Wednesday she never dreamed of having her picture at the Washington museum.
"Of all the dreams that I had, I didn't even know there was a national gallery," she said at the unveiling event, referring to her childhood growing up in poverty, after seeing the portrait for the first time.
"I am living and breathing God's dream for me this day."
The portrait, painted by Chicago-based artist Shawn Michael Warren, depicts Winfrey in a flowing purple gown standing outdoors by a tree.
Her portrait joins that of other famous Americans, including abolitionist Harriet Tubman, Presidents Abraham Lincoln, John F Kennedy and Barack Obama, and former first lady Michelle Obama.
