Dufner shot a second successive seven-under-par round of 65 for a tournament record 36-hole total of 14 under par.
Rickie Fowler shot six under for the day to move third and within one stroke of fellow American Daniel Summerhays, with Jordan Spieth still on six under.
Scotland's Martin Laird is a stroke further back at five under par.
Dufner, who began his second round on the 10th, holed from 176 yards at 18 as his approach to the back of the green rolled down the slope and into the cup for an eagle.
"We got two more rounds to go. We'll see how the course changes, if the wind picks up, gets running fast, it plays different. We'll just see," said the 40-year-old.
World number six Spieth was one shot off the lead after an opening round of 66, but three bogeys on the front nine saw him drop to tied fourth.
Meanwhile, world number one Dustin Johnson missed the cut at eight over par.
American Jason Dufner will take a five-shot lead into the third round of the Memorial Tournament after carding an eagle on the 18th hole on Friday.