Marquez (Repsol Honda) went off the track with 10 laps of the Australian MotoGP to go after starting the race strongly, but the Spaniard's accident allowed Rossi (Movistar Yamaha) to take the chequered flag for the first time since the San Marino MotoGP last month.
Rossi - on his 250th MotoGP race - led home team-mate Jorge Lorenzo by almost 11 seconds, while Monster Yamaha Tech's Bradley Smith claimed third.
Ducati's Andrea Dovizioso and Hector Barbera of Avintia Racing completed the top five.
The victory strengthened Rossi's hold on second spot in the overall MotoGP standings on 255 points, with Marquez (312) well clear and Lorenzo (247) in third.
In all, nine riders failed to finish the race in Australia, with Cal Crutchlow (Ducati) crashing on the last lap when in second spot.