A novel method of aqueous two-phase system (ATPS)-based biphasic recognition chiral extraction has been developed to separate phenylsuccinic acid (PSA) enantiomers. The phase behaviors of ethanol/ammonium sulfate ATPS including phase diagrams, phase volume ratios and phase compositions were studied thoroughly. Based on crucial phase transition points, a modified phase diagram was established experimentally. It was found the phase volume ratios of the system change curves in two parts of ATPS area showed opposite trends, and ideal region with appropriate phase volume ratios and high stability was selected for enantioseparation process. Furthermore, the optimum phase composition for improved enantioseparation was obtained based on two different chiral selector-oriented recognition regions. Then the main system parameters including pH, types of chiral selectors, operational temperature, as well as the PSA enantiomers concentration were also investigated. The maximum separation factor of 4.06 was obtained under the optimized experimental conditions with L-(+)-Tartaric acid diisopropyl ester and Hydroxypropyl-beta-cyclodextrin as the biphasic chiral selectors. It is foreseeable that the ATPS-based biphasic recognition chiral extraction could be beneficial to developing efficient enantioseparation methodologies for other racemic compounds. (C) 2015 Elsevier Ltd. All rights reserved.