After the UMP-ADH-Sephsrose 4B column was made as the affinity manterial,human erythrocyte pyrimidine-5′-nucleotidase(P5′N EC 3.1.3.5.) was purified from the blood of normal subjects by a combination of DEAE-cellulose chromatography、ammonium sulfate fractionation、ion-exchange and affinity chromatography. The results show that the P5′N can adhere to the UMP-ADH-Sephsrose 4B column tightly with high specificity. Polyacrylamide electrophoresis of the purified material shows one strong protein band. The enzyme has pI of 5.2 and a relative molecular mass of 28 000 by polyacrylamide electrophoresis.The anyibody was obtained after the rabbits were immunized with the purified enzyme. It might play an important role in clinical study on hereditary or acquired erythrocyte pyrimidine-5′-nucleotidase dificiency.