To utilize amino acids from food waste as an energy source, l-proline/O2 biofuel cell was constructed using a stable enzyme from hyperthermophilic archaeon for long-term operation. On the anode, the electrocatalytic oxidation of l-proline by l-proline dehydrogenase from Aeropyrum pernix was observed in the presence of ferrocenecarboxylic acid as mediator. On the cathode, electrocatalytic oxygen reduction was detected. Ketjenblack modification of carbon cloth substrate increased the current density due to increased laccase loading and enhanced electron transfer reaction. The biofuel cell using these electrodes achieved a current density of 6.00 µA/cm2. We successfully constructed the first biofuel cell that generates power from l-proline.