− Enzyme fuel cells were operated with cells composed of enzyme anode and PEMFC cathode. Enzyme anodes was fabricated by compression of a mixture of graphite particle, glucose oxidase(Gox) as a enzyme and ferrocene as a redox mediator, and then coated with Nafion ionomer solution. Performances of enzyme unit cell were measured with variation of anode manufacture factors, to find optimum condition of enzyme anode. Optimum pressure was 8.89MPa for enzyme anode pressing process. Highest power density was obtained at 60% graphite composition in enzyme anode. Optimum glucose concentration was 1.7 mol/l in anode substrate solution. The enzyme anode was stabilized by two times of deeping in Nafion solution for 1 sec.