A membrane that can effectively separate the specific isomer only and can maintain a high separation efficiency for a long time by molecular-imprinting a specific optical isomer to be separated such as (D)-serine on an active layer of the composite film in a preparation process of a nano-composite film with stable physical and chemical properties and excellent transmitting characteristics is provided. A method of preparing a polymer membrane for separating optical isomers comprises imprinting optically active compounds on a porous support, thereby forming a molecular imprinted layer on the porous support to separate optionally an optical isomer. A method of preparing a polymer membrane for separating optical isomers comprises the steps of: (a) preparing a porous support; (b) impregnating the porous support with an aqueous mixture solution of an amine monomer and an optically active compounds for molecular imprinting; (c) removing an aqueous solution presenting on a surface of the impregnated porous support; (d) impregnating the porous support of the step(c) with a solvent into which acyl halide is dissolved, and performing interfacial polymerization to form a polyamide layer; and (e) extracting optically active compounds from the polyamide layer, and molecular-imprinting the optically active compounds.
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