Aqueous two-phase flotation (ATPF), which has the advantages of both solvent sublation and aqueous two-phase extraction, is an effective separation technique for separating strongly polar compounds. A highly effective, economically applicable and environmentally safe method has been successfully developed for separating puerarin from Puerariae extract. In an ATPF system composed of polyethylene glycol (PEG)/ammonium sulfate, the effects of various solution pHs, polymer molar masses, concentrations of ammonium sulfate in aqueous solution, nitrogen flow rates, flotation time and initial volumes of the PEG phase were investigated. Under the selected optimal conditions, the separation efficiency of ATPF was more than 87%. The comparison results between aqueous two-phase extraction (ATPE) and ATPF showed that ATPF can effectively separate puerarin from a Puerariae extract with high concentration coefficient. At the same time, the flotation product was purified by high-speed counter-current chromatography (HSCCC), and the purity of the final product was more than 95%. The experimental results show that ATPF-HSCCC is an effective method for preparing higher purity puerarin from a Puerariae extract. (C) 2010 Elsevier B.V. All rights reserved.