The purification and preparation of undegraded prothrombin from bovine plasma was studied with various methods. The more simple and reproducible method for the preparation of undegraded prothrombin is reported in this paper and highly active prothrombin was obtained in good recovery by DEAE-Sephadex column chromatography following ammonium sulfate precipitation. The prothrombin thus purified showed a single band on the disc electrophoresis.