By “interfacial affinity chromatography” on GDX101 column, commercial Candida rugosa lipase (CRL) is fractionated into four fractions containing three isoenzymes (CRL-1, CRL-2 and CRL-3). They have different enantioselectivity for the asymmetric hydrolysis of (R, S)-Naproxen methyl ester in the aqueous-organic solvent biphase system. As analyzed on SDS -polyacrylamide gel electrophoresis, isoelectric focusing and organic solvent treatment, slight structural difference among these isoenzymes has been found. CRL-1 and CRL-2 are associated noncovalently with low molecular mass acidic components in different degree, CRL-3 is disassociated with the low molecular mass acidic components. Because of the perhaps hamper of the low molecular mass acidic components, the open extent of hydrophobic pocket around active site on CRL-3 is bigger than on CRL-1 and CRL-2. According to the slight structural difference, isoenzymes with different enantioselectivity for hydrolysis of Naproxen ester have been immobilized selectively on different hydrophobic supports (GDX101 and YWG-NH₂). Via a sample and easily performed selectivity adsorption step, the purification can be combined with the immobilization. This new method seems to be very suitable for an easily separation of such isoenzymes with slightly different structure.