Flight QF41, an Airbus A330, pushed back from the Sydney terminal at at 3:41pm local time, and lifted into the air at 4:02pm, bound for Jakarta, Indonesia.
More than 70,000 passengers were stranded when Qantas CEO, Alan Joyce, took the drastic step of grounding his entire fleet on Saturday in an attempt to end a long-running industrial dispute with unions.
At the government's instigation, Australia's labour tribunal ordered Qantas to resume flights and banned trade unions, which have waged a damaging campaign of industrial action, from staging more strikes.
Mr Joyce has apologised for the disruptions and vowed to return to "business as usual" over the next 24 hours.
"We very much regret the inconvenience and stress that has been caused by this action. We will be doing all that we can to put things right. And my message today is that all of our customers now can book with confidence with the airline going forward, because all industrial action now ceases," Mr Joyce said.
Australian Prime Minister, Julia Gillard, has expressed her satisfaction that the tribunal, Fair Work Australia, was successful in forcing Qantas and its unions to get the planes back in the air.
"Earlier this morning, Fair Work Australia granted the government's application to terminate all industrial action relating to Qantas. This is a win for passengers stranded around the country and around the world. This is a win for the 1 million Australians who work in tourism," she said.
But Gillard was critical of Qantas' decision to ground its entire fleet.
"I do not accept that Qantas' only choice on Saturday was to take the extreme action of grounding all planes and leaving tens of thousands of passengers stranded. I do not accept that," she said.
Qantas says it has lost about A$70 million (£46 million) since September from industrial action in its dispute with three trade unions over pay, working conditions and its Asian plan.
Full operations are not expected to be restored until Wednesday.
