To provide a method for carrying out the separation and determination of α-amylase isozyme, especially the method for carrying out the separation and determination of the α-amylase isozyme by using an enzyme sensor comprising an electrode formation obtained by combining a hydrogen peroxide electrode with a specific immobilized enzyme membrane.

This method for carrying out separation and determination of α-amylase isozyme, which can separately determining the amylase activities derived from the salivary gland and the amylase activities derived from the spleen comprises measuring of the whole amylase activities of a biological fluid sample and remaining amylase activities of the biological fluid sample obtained by specifically inhibiting the amylase derived from the salivary gland in the biological fluid by a monoclonal antibody, by using an enzyme sensor having an electrode construction obtained by laminating an immobilized glucose oxidase enzyme membrane, the first limiting membrane, an immobilized α- glucosidase enzyme membrane and the second limiting membrane on a hydrogen peroxide electrode one by one, to measure the amylase activities derived from the salivary gland and the amylase activities derived from the spleen.