The opposition got closer, but the former Ducati star also made minor improvements throughout the session.
The 2007 world champion's superiority was never threatened and Stoner enjoyed a 0.610sec advantage by the end of the 45-minutes. Stoner's third lap would still have been quick enough to keep him fastest and he was the only rider to lap in the 1min 55sec, with his best a 1min 55.752sec. Stoner had set a 1min 55.681sec during Monday's test.
In a repeat of the Qatar test, Stoner's nearest rival once again proved to be team-mate and 2010 title runner-up Dani Pedrosa.
Hector Barbera produced one of the biggest surprises of the evening by snatching third, after benefiting from a tow behind fellow Ducati rider Nicky Hayden.
Fourth place went to San Carlo Honda Gresini rider Marco Simoncelli, but it was another Italian who grabbed greater attention in the closing minutes.
MotoGP megastar Valentino Rossi, just 13th at the end of testing, finished his first official grand prix session with a Ducati Desmosedici in a far more competitive fifth place - ahead of his former Factory Yamaha Team riders. Rossi, winner of last year's race for Yamaha, worked his way steadily forwards from the lower reaches of the top ten to fifth (+0.727sec from Stoner) on his 19th and final lap.
Then came the factory M1s of Ben Spies and Yamaha's reigning world champion Jorge Lorenzo, with third Repsol Honda rider Andrea Dovizioso, new Pramac Ducati rider Randy de Puniet and Monster Yamaha Tech 3's Colin Edwards completing the top ten.
Rossi's team-mate Hayden was eleventh and Simoncelli's team-mate Hiroshi Aoyama twelfth, although Pedrosa-to-Aoyama was separated by just 0.625sec.
de Puniet's team-mate Loris Capirossi was 13th, while Cal Crutchlow's first grand prix track session in any class ended with the Monster Yamaha Tech 3 rookie in 14th.
The Englishman, who lost the tip of his left little finger in a crash during testing on Monday, was 1.677sec slower than Stoner (but only a second from Pedrosa).
Fellow rookie Karel Abraham (Ducati), the lone Suzuki of Alvaro Bautista and struggling Moto2 champion Toni Elias (Honda) completed the 17 rider list.
The unique four-night race weekend schedule for the Qatar event will see two further MotoGP free practice sessions on Friday, followed by qualifying on Saturday, then warm-up and the race on Sunday.