The smooth-swinging South African recovered from bogeys on the first two holes to card a six-under-par 66 at the rain-softened Redstone Golf Club in the final US Tour event before next week's Masters.
Oosthuizen, who romped to a seven-stroke victory at the 2010 Open, launched his late surge with four consecutive birdies from the 12th to post a 17-under total of 199.
American Hunter Mahan was alone in second place after carding a 65, with Swede Carl Pettersson (67) and Britain's Brian Davis (69) a further stroke back at 14 under.
For the second round in a row at Redstone, players were permitted to lift, clean and replace their balls on a course that had been saturated by more than an inch of rain on Thursday.
"I had to compose myself," Oosthuizen said, referring to his bogey-bogey start. "Other than that, I hit it well and started making a few putts on the back nine.
"I was driving it well, my iron play the whole week has been really good and I just made some good putts today.
"The greens here are really fast, which I enjoy. If it's not windy here, you feel like there are a lot of birdies out there."
Asked how he would approach Sunday's final round, Oostuizen replied with a smile: "Pretty much the same as today, try and not start bogey-bogey.
"It's always a good opportunity going in with a lead. I think it's going to be good fun tomorrow."
Defending champion Phil Mickelson was among a group of three players tied for sixth at 11 under after shooting a three-birdie 70.
Three-times Major winner Ernie Els, who needs to win the Houston Open to qualify for next week's Masters, was at eight-under 209 after shooting a second successive 69.
"I've just got to stick to what I'm doing," said the South African former world number one. "I'm playing really well.
"Obviously, I would love to get a win. I'd like to play a solid round of golf tomorrow. If it really gets going, that will be really a plus."
Earlier in the day, American Jeff Maggert birdied three of his last eight holes to grab a one-shot lead when the weather-delayed second round was finally completed at Redstone to get the tournament back on track.
The 48-year-old went on to shoot 76 in the third round to finish a long day tied for 14th at eight under par.
Maggert, a three-times winner on the US Tour, is playing his ninth and final event this year on a major medical exemption following shoulder surgery and needs $202,609 to retain his status for next season.