Ionic liquids-based aqueous two-phase system (ILs-ATPS) offers an alternative approach for the extraction of tetracycline (TC) through their partitioning between two phases. The procedure was composed of 1-butyl-3-methylimidazolium halide ([Bmim]X(X = Cl, Br)) and K2HPO4 using a liquid-liquid extraction technique. The influence factors on partition behavior of TC were optimized systematically, including the quality of salts and IL, the amount of TC, pH value, and temperature. The maximum extraction efficiency of TC could reach 99%. The interactions between the ATPS and TC have been examined by isothermal titration calorimetry. Extraction of antibiotics TC using ILs-ATPS could achieve nearly complete extractions in a single-step by way of the proper tailoring of their concentrations in the aqueous media.