A method of producing alcohol oxidase wherein Hansenula polymorpha yeast is cultivated in a nutrient broth containing inorganic nutrient salts of which either the phosphate, nitrate or potassium is present in a cell-growth-limiting concentration at a temperature of 25.degree. to 45.degree. C. and a pH of 4.0 to 6.0 with oxygen being supplied as air for a mixture of air and oxygen. According to the invention, an organic substrate consisting of glycerin, sorbite or xylose, noncatabolite-repressive to alcohol-oxidase is added to the broth in a concentration of 0.1 to 1.0% in a nutrient solution at a rate of 0.03 to 0.15 .sup.-1.