The Alachua County Sheriff's Office is the chief law enforcement agency for unincorporated areas of Alachua County, Florida, USA. The current sheriff, Sadie Darnell, is the first female sheriff for the county. The agency has its headquarters in Gainesville, Florida and serves a population of 217,955 within 970 square miles.

The agency performs law enforcement functions, operates a SWAT team, runs the county jail, is responsible for court security, and performs other various law enforcement related tasks for the county.

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The Alachua County Sheriff's Office was founded prior to the US Civil War in 1824; the same year as the founding of Alachua County, Florida. This is prior to the establishment of statehood for Florida in 1845.