Abstract: A novel polyol oxidase is derived from a microorganism belonging to the genus Penicillium and is specified by the properties (a) to (e): (a) it has a stable pH of 6.0 or higher and an optimum reaction pH of from 7.0 to 9.0; (b) it has an operating temperature of 50° C. or lower and an optimum reaction temperature of 40° C.; (c) it has a molecular weight of about 113 kDa; (d) it specifically recognizes the structure of a polyol in which the OH groups at positions 2 and 3 are in the L-erythro configuration and reacts with the polyol, but cannot recognize the structure of a polyol in which the OH group at position 4 is in the L-ribo configuration and does not react with the polyol; and (e) it has substrate specificity for D-arabitol, erythritol, D-mannitol, and D-sorbitol, which are listed in descending order of specificity.