A novel transformant-based glucose biosensor is proposed using glucose oxidase (GOD) as the molecular recognition material in order to solve the problem of deactivation of the enzyme with time. In this paper, the feasibility of this approach for a glucose biosensor is demonstrated as a preliminary study. A. niger is used as a source of the GOD gene. The yeast strain P. pastoris X-33 is used as the host for plasmid construction, cloning, and enzyme expression. The yeast transformant-based glucose 131biosensor consists of a ring filter, disc housings, and a flat electrode (30 mm diameter, 8.5 mm thick). A reaction chamber with an inner volume of 300 μl is constructed by sandwiching a ring filter made of porous sintered metal between two disc housings. Both the GOD protein and its transformant are enclosed in the reaction chamber. Our results indicated that (i) the GOD gene transformant, which is capable of not only secretory expression but also constitutive expression, was obtained, and (ii) deactivated GOD, with time, can be replaced with newly produced recombinant GOD from the GOD gene transformant.