Characteristics of a novel method of α-amylase activity determination were investigated. The Principles of the present method were based on the light absorbance change through a starch-agar gel made in a microplate well by α-amylase action. The sensitivities of β-amylase and glucoamylase with this method were less than 2% to the sensitivity of α-amylase. α-amylase activity in a extract from rice koji and Rhizopus culture liquid were successfully determined and glucoamylase was not affected to determine α-amylase activity as a result. Since the present method can be possible to determine α-amylase activity selectively, this method was considered as an effective for quick and multi-sample treatment by an automatic devise.