Purification of plant-esterase from flour in an aqueous two-phase system (ATPS) was investigated. The effects of various process parameters such as the type of aqueous two-phase systems, the phase-forming salt, the molecular weight and concentration of PEG, the system pH, and the types and concentrations of neutral salts on partitioning of plant-esterase were evaluated. Optimized conditions for the purification of plant-esterase were found in polymer-salt systems, with especially promising results in the PEG1000/NaH2PO4 system. Using 27.0% PEG1000/13.0% NaH2PO4 (w/w, pH 5.0), and 27.0% PEG 1000/13.0% NaH2PO4/6.0% (NH4)(2)SO4 (w/w. PH 5.0), plant-esterase was purified by a two-step extraction. Compared to the results obtained with the conventional salting-out method, this method had a comparable yield (83.16% versus the original yield of 80%), but produced plant-esterase that was 4.8 times as pure (18.46 fold). Integrating dialysis into the aqueous two-phase extraction removed (NH4)(2)SO4 from the purified plant-esterase. Finally, plant-esterase was freeze-dried to convert the product to powder. This work offers a simple and more efficient process to purify and concentrate plant-esterase. Plant-esterase is used in applications such as organophosphorus compounds (OPs) detection and since our method makes this enzyme easier to isolate, it will enhance researchers' ability to explore these applications. (C) 2010 Elsevier Ltd. All rights reserved.