Leading by two shots overnight, Stefani followed his opening round 65 with a one-under-par 70 at the Copperhead layout at Innisbrook Resort to reach the halfway stage at seven-under.
The 31-year-old briefly lost his lead but regained it when he drained a 14-foot birdie putt on the 15th hole.
"The lead is one thing, but leading on the weekend is another," Stefani told reporters.
"I wasn't any more nervous than I am any round. I'm always a little bit excited about playing and I get a little nervous, which I enjoy."
Stefani finished the day in front but his lead was cut in half as Australia's Adam Scott and South Korea's KJ Choi both moved to six under.
Scott shot a flawless 66 while Choi, already a double winner of the Tampa Bay Championship, carded a 67, including an eagle-three at the first hole.
"To go bogey-free anywhere is good, and I think (to do it) here is particularly good," Scott said.
"It's quite a tricky golf course, and mistakes are easily made."
Scott finished third behind Tiger Woods at last week's Honda Classic and is back in form after his meltdown at last year's British Open.
With victory seemingly in his grasp, Scott bogeyed the last four holes to allow South Africa's Ernie Els to slip past him and win by a stroke.
Scott and Els remain close friends and Scott revealed that the pair made a flying trip to Augusta together at the start of a week for a practice round before the Masters.
"Whether he won it, or I helped him win it a little bit - it doesn't matter. He won it," Scott said. "It probably eased the pain a little bit that he's a close friend of mine, and I could feel some happiness for him."
Four players - Brian Harman, Harris English, Jason Dufner and Peter Tomasulo - finished tied for fourth at minus five. Dufner matched Scott's 66 while Harman and English both bogeyed their final holes to miss out on the chance to finish higher.
Spain's Sergio Garcia shot a 67 to finish within three shots of the lead while the defending champion, Britain's Luke Donald, shot a 72 to finish at even par, alongside U.S. Open champion Webb Simpson. (Reporting by Julian Linden in New York; Editing by John O'Brien)