\Diaphorase and 2-amino-3-carboxy-1,4-naphthoquinone as an enzyme and a mediator for NADH oxidation, respectively, were successfully co-immobilized on a glassy carbon electrode with poly-L-lysine and glutaraldehyde as a polymer support and a cross-linking reagent, respectively. The diffusion-controlled oxidation of NADH (10 mM) was achieved around -0.25V vs Ag\AgCl\KCl(sat.).