Residents were warned to stay indoors as more than 100 deputies and officers from four law enforcement agencies searched for the gunman after the confrontation at a home in the Ocala National Forest.
The suspect, 29-year-old Jason Lee Wheeler, escaped on a motorcycle, which was later found ditched, said Nick Pallitto, a spokesman for the Lake County Sheriff's Office.
Deputy Wayne Koester died at a hospital, Pallitto said. The two other deputies were being treated at a hospital for leg wounds.
Two nearby schools were on lockdown, and Florida Highway Patrol troopers closed portions of two roads, authorities said.
Some of the deputies, armed with shotguns, stopped cars passing through the area.
"It's an intensive manhunt, and we're going to get this guy," said Pallitto.