The Australian, who now has 38 career wins, finished 22.077 seconds ahead of his Japanese team-mate Tadayuki Okada and 22.471sec in front of the Italian, Luca Cadalora.
Okada, who was an uncertain starter after injuring his left hip in Saturday's final qualifying, capitalised on an error by his Spanish team-mate Alex Criville on the penultimate lap. Criville, who now trails Doohan in the standings by 31 points, was second for most of the 28-lap race but went off the track after a mistake and finished fifth.
Olivier Jacque of France won the 250cc race after overtaking Germany's Ralf Waldmann on the final lap. The world champion, Max Biaggi of Italy, extended his lead in the standings with third place.
The Japanese Honda rider Noburu Ueda took the lead in the 125cc standings from Aprilia's Valentino Rossi, winning in 40min 19.719sec, just 0.004sec ahead of the Italian.
In the 90th anniversary Isle of Man TT races, Ulsterman Phillip McCallen is looking to repeat his runaway success in yesterday's opening event, the Formula One.
The big test will come in today's four-lap lightweight TT, a race the 33-year-old Honda rider wants to win more than any other. Twice he has run out of fuel when leading, and last year a cracked exhaust dropped him from first to fourth.Reuse content