α-glycerophosphate oxidase is produced by cultivating microorganisms belonging to genus Pediococcus, Streptococcus, Lactobacillus or Leuconostoc in a nutrient medium containing at least one compound selected from the group consisting of α-keto acids represented by the formula,
R--COCOOH
wherein R is CH.sub.3 (CH.sub.2).sub.m --, HOOC(CH.sub.2).sub.n -- or CH.sub.2 (OH)CH(OH)CH(OH)CH-- (in which m is an integer of 1 to 3 and n is an integer of 0 to 2) and salts thereof, and then α-glycerophosphate oxidase is recovered from the resulting culture broth. α-ketobutyric acid, α-ketovaleric acid, α-ketocaproic acid, α-ketomalonic acid, oxalacetic acid, α-ketoglutaric acid and α-ketogluconic acid are disclosed.