The invention discloses a method utilizing enzyme measure to separate a mixed rotating selenium-methyl selenocysteine to prepare an optical pure selenium-methyl selenocysteine. The method acerylates the mixed rotating selenium-methyl selenocysteine with erhyl anhydride in sodium hydroxide solution and acidificates with hydrochloride to achieve a mix N ethyl acyl selenium methyl selenocysteine. Then, in certain PH value, reaction temperature, enzyme amount, substrate and cobalt ion concentration, the mix N ethyl acyl selenium methyl selenocysteine is separated by acylate enzyme I of swine renalto obtain an oprical pure L selenium methyl selenocysteine and an N ethyl D selenium methyl selenocysteine. And finally the N ethyl D selenium methyl selenocysteine is heated and hydrolyzed with hydrochloride and is neutralized with ammonia water to obtain an optical pure D selenium methyl selenocysteine. The method has the advantages of convenient for operating and low in cost, which is suitablefor industrialized production of the optical pire selenium methyl slenocysteine.