Middlesboro, Kentucky. Not to be confused with Middletown, Kentucky, near Louisville. Middlesboro (local /ˈmɪdəlzbɜːrə/) is a home rule-class city in Bell County, Kentucky, United States. The population was 10,334 at the 2010 U.S. census, while its micropolitan area had a population of 69,060. It is located 1 mile west of the Cumberland Gap and is the largest city in southeastern Kentucky.