The store, located in Sevierville, is the first business to open at Exit 407: Gateway to Adventure - a 200-acre development owned and operated by the Eastern Band of Cherokee Indians.
The new site spans 74,000 square feet, boasts 120 gas pumps, a 250-foot car wash and has 400 full-time employees.
Fans reportedly began lining the streets on Sunday evening ahead of the opening at 6AM on Monday morning.
Local traffic officials previously warned that any new Buc-ee's stores tend to attract a 'miles-long line of cars.'