The 69-year-old country star made the declaration following a story in the National Enquirer that she had been rushed to hospital.
"There is absolutely no truth at all that I have stomach cancer," the statement on her website said.
"It is true that I had kidney stones," she said. "I had them removed three weeks ago and I am doing just fine!"
Parton added that she was already back at work following the operation.
"I am back to work and last week I was at Dollywood filming parts for my new movie Coat of Many Colors," she wrote. "I love and appreciate everyone's concern."
Fears for Parton's health were sparked when The Eagles star Don Henley said she had overcome a "medical issue" to shoot a video with him in Los Angeles.
Speaking during an interview at the Americana Music Festival last month, the musician said Parton had "got out of the hospital bed" to join him in the video for When I Stop Dreaming.
Singer Dolly Parton has issued a statement denying a report that claimed she has stomach cancer.