Ionic liquids-based aqueous two-phase extraction (ILs-ATPE) offers an alternative approach to the extraction of tetracycline (TC) through their partitioning between two phases. Single-stage and multi-stage strategies have been evaluated and compared for the purification of TC using ATPE composed of 1-butyl-3-methylimidazolium halide ([Bmim]X(X=Cl,Br)) and K2HPO4. The influence factors on single-stage extraction behavior of TC were optimized systematically, including the pH value, tie line length, and volume ratio. The optimal extraction efficiency of TC could reach above 95% when the volume ratio is higher than 1.5 and the tie line length is 30.52%. The multi-stage ATPE was also investigated by simulating a three-stage crosscurrent operation in test tubes. According to the TC isotherm curve and respective McCabe?Thiele diagrams, a predicted optimized scheme of the countercurrent multi-stage ATPE was determined. TC can be purified in the IL-rich top phase with a final extraction efficiency of 99% and a final TC concentration of 0.25 mg/mL, if a three- -stage [Bmim]Cl-K2HPO4 ATPE with volume ratio of 0.5 and tie line length of 30.52% was employed. Thus, the multi-stage extraction with small volume ratio is necessary to achieve a higher recovery yield, resulting in the reduction of the IL consumption.