Cycling Australia confirmed on Twitter that O'Donnell had "set a new #UCIHourRecord of 46.882km" for the gruelling velodrome event.
"I can't believe I broke a world record," the 41-year-old said after getting off her bike and hoisting it above her head at the Super-Drome in Adelaide.
The previous record for distance ridden in 60 minutes by a woman was 46.274 kilometres set by American Molly Shaffer Van Houweling in Mexico in September.
O'Donnell said she had been nervous the day before the event, but had felt a lot more "calm and ready" as she prepared to race on Friday.
She kept to her plans for pacing herself for the sprint against the clock, and said she focused on what she was doing despite the pain.
"How you feel is kind of irrelevant, you just have to have confidence in the plan," she said.
O'Donnell, a doctor, has represented Australia at three UCI Road World Championships but said it was a matter of finding the right event for her.
"I wanted to do something great," she said, adding that she felt the record was a good start.
Ahead of the event, she said that her bid was as much psychological as physical.
"It's about planning and preparation and logistics and training and then certainly enacting the plan the best way you can," she told the Australian Broadcasting Corporation.
Van Houweling was among the first to congratulate the Australian, tweeting: "Very impressive new #UCIHourRecord".