Enzyme biofuel cells utilizing glucose dehydrogenase as an anode enzyme were constructed. The glucose dehydrogenase is composed of a catalytic subunit, an electron transfer subunit, and a chaperon-like subunit. Cells, constructed using either a glucose dehydrogenase catalytic subunit or a glucose dehydrogenase complex, displayed power outputs that were dependent on the glucose concentration. The catalytic subunit in the anode maintained its catalytic activity for 24 h of operation. The biofuel cell which composed of glucose dehydrogenase complex functioned successfully even in the absence of an electron mediator at the anode cell. These results indicate the potential application of this thermostable glucose dehydrogenase for the construction of a compartment-less biofuel cell.