The laid-back American, who won his second title on the US circuit at the Byron Nelson Championship on Sunday, fired a flawless six-under-par 64 on another hot and breezy day at the iconic Colonial Country Club.
"I've been really, really confident out there, playing some nice golf," Dufner said after reaching 27 of 36 greens in regulation over the first two rounds. "I just feel really comfortable with my game.
"I've said it now for a couple of months, when I step to the first tee I feel like I'm going to play a good round of golf. That's a nice way to play golf. It's a comfortable way to play golf."
Dufner covered his last nine holes in a sizzling four-under 31 to post an 11-under total of 129, two better than overnight leader and 2010 champion Zach Johnson, who carded a 67.
Fellow Americans Bo Van Pelt (64) and Tommy Gainey (67) were a further two strokes back at seven under, one better than compatriot Tom Gillis (69).
Dufner, who needed 164 US PGA Tour starts before clinching his maiden victory at the New Orleans Classic four weeks ago, continued where he left off on Thursday after shooting a 65.
A stroke behind the pacesetting Johnson overnight, the 35-year-old birdied the 13th and 17th to take over at the top after teeing off at the par-four 10th.
Dufner then picked up further shots at the first, second, third and fifth, where he rolled in an 18-foot putt, to cement his status as the hottest player in the game.
Should he triumph on Sunday, he would become only the second player (after Ben Hogan in 1946) to win the Colonial Invitational and Byron Nelson Championship in the same year.
"It would be a great accomplishment to do that," Dufner said. "After yesterday I felt pretty good about my chances of playing well this week.
"So we will just go into the weekend with the lead ... hopefully continue to play some good golf and maybe make a little bit of history."
Rickie Fowler, who clinched his maiden US PGA Tour victory at the Wells Fargo Championship three weeks ago, summed up Dufner's dominance after playing with his fellow American in the first two rounds at Colonial.
"We're going to have to play catch-up this weekend," smiled Fowler after shooting a second successive 68 to finish seven strokes behind Dufner.
Johnson, who led by one shot after the opening round, was happy with his 67 after having to contend with gusting winds in the afternoon.
"Today, it was good," the 2007 Masters champion said after mixing an eagle at the first with four birdies and three bogeys.
"I got off to a great start ... hit a couple of bumps along the way. I finished strong.
"I got some momentum going on the back (nine) but it seems like every time I made a birdie I put myself in a pretty difficult predicament to make par. I'm excited about tomorrow."
The cut fell at three-over 143 with defending champion David Toms, US Ryder Cup captain Davis Love III and exciting Japanese talent Ryo Ishikawa among those failing to advance.