The method of PEG/phosphate salt two phase extraction as the first step of purification to prepare duck serum choline esterase was first used in this paper.The procedure was not only simple,rapid but also high in the activity recovery of the choline esterase.The purified choline esterase with specific activity 279.9 U/mg was followed by DEAE-sephadex A50 and sephadex G200 chromatography.The choline esterase was purified 1018-fold and the activity recovery of 43.4 per cent was obtained.Studies on the properties of the choline esterase showed that it was a kind of glycoproteins and acid hydrolases. The isoelectric point of 4.2 and the optimum pH of 7.5 were obtained. The K_m of 9.8×10^-5mol/L with butyryl-thiocholine iodide was determined. SDS and polyacrylamide gel electrophoresis showed that the choline esterase existed in the different polymers composed of the same subunit. The molecular weight of subunit was 78000, and the subunit had the activity of the whole choline esterase.