The locomotive steam engine made the journey from London King's Cross to York, where it will go on display at the National Railway Museum (NRM).
It arrived in York almost an hour later than expected, after it was twice held up by reports of trespassers.
The engine, which retired from service in 1963, has been restored for the NRM, which bought the locomotive for 2.3 million pounds in 2004.
Former MP turned TV presenter Michael Portillo, said: "This is certainly the most famous journey and most famous locomotive in Britain."
More From This Section
The first disruption on its inaugural run happened near St Neots when the train was brought to a standstill while British Transport Police cleared the lines after reports of around 60 people on the track. No arrests were made.
Network Rail said the number of people on the trackside had caused "safety concerns" and resulted in a number of services, including Flying Scotsman, having to slow down."
Crowds gathered at King's Cross for its scheduled departure at 07:40 and thousands lined the route, while some of the 297 passengers paid up to 450 pounds for a ticket to travel on the train.
Among the passengers was 83-year-old Ron Kennedy, from Leigh-on-Sea, in Essex, who drove Flying Scotsman from 1956 until it was retired in 1963.
He said "It's unbelievable. I never dreamt about being on it again. To be out with it is just fantastic."
The locomotive undertook a series of test runs through Cumbria and Lancashire earlier this year.