The British-based singer taped the interview via satellite from London, for airing in America tomorrow.
It’s the first time she has spoken publicly in depth about the adoption.
According to a member of Oprah's audience, Madonna said that she was startled by reports quoting the toddler's father, Yohane Banda, as saying he did not realise he was signing away custody of his son, David, "for good".
"She said she met with the father, she looked him in the eye," audience member Sheryl Lewis recounted.
Madonna, she added, said she acted according to the law and had both "oral and written approval ... and now the press have gotten to him", Ms Lewis said.
In an interview posted on Time magazine's website, Banda said that for his son's sake he would not contest the adoption.
"I don't want my child, who is already gone, to come back," he said. "I will be killing his future if I accept that."