Publisher Summary Glucose oxidase catalyzes the oxidation of D-glucose to δ-D-gluconolactone and H 2 O 2 in the presence of molecular oxygen. In a subsequent step δ -D-gluconolactone is nonenzymatically hydrolyzed to D-gluconic acid. This enzyme has been demonstrated in various Aspergillus and Penicillium species and has recently been purified from Phanerochaete chrysosporium . This chapter focuses on assay method and purification procedure of the enzyme from Phanerochaete chrysosporium . It discusses the properties of the enzyme. Staining of the purified glucose oxidase from P. chrysosporium for protein-bound carbohydrate by the dansyl hydrazine method shows no detectable carbohydrate, whereas an equal amount of commercially prepared glucose oxidase from Aspergillus niger , which is known to be a glycoprotein, stains positive. This finding is of interest that a majority of peroxisomal proteins appear not to be glycosylated and that H 2 O 2 production in ligninolytic cultures of P. chrysosporium , presumed to be due to glucose oxidase activity, has been shown to be localized in periplasmic, peroxisome-like structures.