Pyranose oxidase (EC 1.1.3.10 ; PROD) catalyzes the oxidation of aldopyranoses at the position C‒2to yield the corresponding 2‒keto-aldoses and H2O2 , using oxygen as an electron acceptor. The enzyme shows broad substrate specificity as well as reactivity for 1,5‒anhydro‒d‒glucitol (1,5‒AG), which is known as a clinical glycemic marker. It is considered that the reactivity of PROD for 1,5‒AG is useful in the development of an amperometric-type biosensor, which is a convenient diagnostic device for selfmonitoringblood glucose (SMBG). However, the levels of dissolved oxygen in blood affect biosensorsystems that are equipped with an artificial electron mediator. In the present study, we attempted to develop an O2‒insensitive oxidase that would improve the dye-mediated dehydrogenase activity. We performed site-directed mutagenesis on PROD isolated from basidiomycetous fungus No. 52, which generated 11 mutants. The amino acid substitution Q421A exhibited a significant decrease (8.8% of wild type) in its oxidase activity, whereas it maintained its dehydrogenase activity (67% of wild type). In this study, we characterized PROD mutants from basidiomycetous fungus No. 52, which showed improved dye-mediated dehydrogenase activity.