By modifying the carbon electrode with a yeast extract (YE) using a support material (SM), a complete bio-anode was established without adding any extrinsic enzymes and mediators in a glucose–air fuel cell. The yeast extract was mixed into a paste with carbon black and an SM, i.e., glutaraldehyde (GA), TritonX-100, polyethyleneglycol, chitosan, or agarose. Chitosan was the best support, producing lower overpotentials and a good stability. Optimization of the paste composition and its loading were carried out for the bio-anode of a glucose–air fuel cell. The fuel cell generated a power of 33 μW cm−2 at 333 K with an aqueous glucose solution without adding any extrinsic enzymes and mediators. It showed about 70% of the initial power output at a stable condition. The bio-anode is expected to be used for energy recovery from hot wastewater-containing glucose.