To provide a method for determining D-alanine capable of measuring a very small amount of D-alanine specifically and in excellent sensitivity.

In a method for determining D-alanine by adding a D-amino acid oxidase to a specimen, generating pyruvic acid, adding NADH and lactate dehydrogenase to pyruvic acid, converting pyruvic acid to lactic acid and NADH to NAD and measuring NADH consumed in a unit time, the lactate oxidase is added to the measurement system and a cycling reaction for converting lactic acid to pyruvic acid is materialized to provide the objective method for determining D-alanine. Consequently, D-alanine can be accurately measured more simply in a shorter time than a conventional HPLC method. D-alanine in body fluid which has been impossible by a conventional enzymatic method can be carried out for the first time.