To design and construct a novel bio-anode electrode based on the oxidation of glutamic acid to produce 2-oxoglutarate, generating two electrons from NADH. Efficient enzyme reaction and electron transfer were observed owing to immobilization of the two enzymes using a mixed self-assembled monolayer. The ratio of the immobilized enzymes was an important factor affecting the efficiency of the system; thus, we quantified the amounts of immobilized enzyme using a quartz crystal microbalance to further evaluate the electrochemical reaction. The electrochemical reaction proceeded efficiently when approximately equimolar amounts of the enzyme were on the electrode. The largest oxidation peak current increase (171 nA) was observed under these conditions. Efficient multi-enzyme reaction on the electrode surface has been achieved which is applicable for biofuel cell application.