AbstractThe present study reports the design of a novel bioanode to deeply oxidize glucose in an enzymatic biofuel cell (EFC). This enzymatic glucose cell utilizes three co‐immobilized enzymes: NAD‐dependent glucose dehydrogenase (GDH), NAD(P)+‐dependent gluconate‐5‐dehydrogenase (Ga5DH), and diaphorase (DI). Glucose is oxidized to gluconate by NAD‐dependent GDH, gaining two electrons per glucose; the gluconate obtained as a by‐product is oxidized at the C5 carbon to 5‐keto‐gluconate by Ga5DH. Operation of our bioanode enabled the oxidation of glucose in two stages, resulting in the gain of four electrons. The three‐enzyme EFC provides a maximum power density of 10.51 ± 1.72 μW cm–2, which is about 1.6 times higher than the maximum power density of an EFC using a bioanode based on the co‐immobilization of two enzymes (GDH and DI). Our results hold promise for increasing the current density of EFCs, and for application in glucose biosensor.