Whincup clocked a fastest lap of one minute and 12.7590 seconds to finish quickest on the day, pulling out the time with just one minute left in the day's final 30-minute practice session.
The TeamVodafone pilot, his Commodore decked out in a camouflage-patterned livery in support of Townsville's military community for this weekend's event, is aiming to dominate the Reid Park street circuit after a disappointing round last time out in Darwin.
Holden's Lee Holdsworth came in second overall with Whincup's team-mate Craig Lowndes in third.
Garth Tander was fourth fastest with Ford's Mark Winterbottom sixth on the standings to be the fastest driver in a Falcon.
Reigning champ James Courtney's woes continued, clocking the day's 19th fastest time overall.
The Townsville 400 gets under way on Saturday with a 200km race following qualifying and top 10 shootout in the morning.