Abstract: By reducing the length of diethylaminoethanol-cellulose and increasing the pH in elution solution,we developed an improved method for purifying glycolate oxidase with high isoelectric value (pI > 10.0) from green leaves of Spinacia oleracea, Brassica parachinensis, and Phaseolus vulgaris. Purified glycolate oxidase showed high specific activity (54.6-197.0 U.mg-1) and high pI (>10.0). We achieved 21.6-122.68-fold purification and 4.1%-71.5% yield from crude solution during this purification procedure. SDS-PAGE of the glycolate oxidases revealed a 40-kD band, which indicates that the subunit molecular weight of glycolate oxidases in the 3 different plants was identical.