Dufner, who let a chance to win a major title get away at last year's PGA Championship, completed 36 holes at the Innisbrook Resort course without suffering a bogey.
His two-day total of 10-under 132 put him in position to capture his first victory on the PGA Tour.
First-round leader Padraig Harrington, who Thursday shot the lowest 18-hole score of his career with a 10-under 61, stumbled to a 73 Friday and was tied for second at 134 with William McGirt.
The group at 135 included Luke Donald, who would vault past Rory McIlroy and reclaim his No. 1 world ranking with a victory this week. Justin Rose, a winner last week in the World Golf Championships event at Doral, was in a tie for 13th at 137.
John Daly, playing in the United States for the first time this year, made the cut with a 1-under 141 total. Daly survived the cut just six times in his 18 PGA Tour appearances last year.
Dufner opened last week's WGC tournament with a 66, but eventually faded to a tie for 29th. His career to this point has been highlighted by his near miss at the PGA last August, when Keegan Bradley rallied to defeat him in a playoff.