The crash occurred about 10:30 EDT Tuesday on Route 67 in Johnson County.
Tennessee Department of Safety spokesman Mike Browning said the fatality was 57-year-old Alicia McCoy of Mountain City.
WJHL-TV in Johnson City reported McCoy was the patient being transported to a hospital by the Johnson County Rescue Squad.
The ambulance driver was thrown out when the vehicle ran into a ditch and overturned after being struck in the front by a sedan. He is 45-year-old Joseph Barlow, who was reported in critical condition Wednesday morning.
Less seriously injured were rescue squad member Kevin Colson of Laurel Bloomery and the driver of the car, Barbara Buchanan of Elizabethton.