Abstract This paper reports the development and optimisation of a glucose/O2 biofuel cell. At the anode, the glucose oxidation was performed by glucose oxidase (GOx) co-immobilised with a phenanthrenequinone mediator. At the cathode, the catalysis of O2 using tetrathiafulvalene and polyphenol oxidase (PPO) has been demonstrated for the first time. The open circuit voltage (OCV) of the optimised biofuel cell reached 0.32V, and the maximum power density achieved was 40.8μWcm−2 at 0.2V. This original new GBFC showed quasi-stability in both OCV and power density for more than 25days.