Sweden's Henrik Stenson was alone in second after firing a seven-birdie 66, with former U.S. Open champion Justin Rose of England a further stroke back at 10 under, also after a 66.
Long-hitting Day, who won his first major title at last year's PGA Championship, was delighted with his form after making a slow start to the PGA Tour season with one top-10 finish in four tournaments.
"I'm trying to make sure that I am deliberate on every pre-shot routine when I am out there," Day told Golf Channel after charging clear of the chasing pack with four birdies in his last seven holes.
"That was the missing part for me earlier in the year, that focus and that kind of killer instinct going into each shot like it's my last ... I am definitely coming around nicely now."
Day, a seven-times winner on the PGA Tour, has previously struggled at Bay Hill where his best finish was a tie for 17th last year.
"Obviously I've hit it better and I have putted great (this week)," said the 28-year-old. "In the past, I have always thought this was a very, very difficult course.
"I haven't had a great record in Florida, Bermuda greens and Bermuda grass have been very difficult for me, but this week it's different.
"We have different grass, over-seeded, which I enjoy chipping off. I enjoy hitting out of the rough and the greens are phenomenal right now. They are rolling true and I love the surface."
World number two Rory McIlroy, shockingly erratic off the tee in his opening 75, improved greatly as he carded a 67 to ensure he would make the cut, which fell at level-par 144.
"I drove the ball horrific yesterday, I haven't driven the ball like that in a long, long time," the Northern Irishman said. "I came out to the range last night and worked on a few things, figured something out.
"The main objective today was just to make it to the weekend, two more opportunities to play and try to figure it out."
(Reporting by Mark Lamport-Stokes in Los Angeles; Editing by Larry Fine)
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