Biofuel cells are an attractive alternative to conventional fuel cells, because they use biological catalysts. We report in this article the construction of an ethanol / O2 enzymatic biofuel cell. In the first time, the performance comparison with different methods for Ethanol/O2 biofuel cell has been study. Alcohol dehydrogenase, a Nicotinamide Adenine Dinucleotide (NAD) dependant enzyme, is immobilized with NAD + , diaphorase and vitamin K3 (VK3) on the electrode. The oxygen is reduced at the cathode with laccase and 2,2'azinobis(3-ethylbenzothiazoline-6-sulfonate) (ABTS).The performances of the electrodes are improved thanks to addition of carbon powder (KS6 and Super P ® carbon). The benefit of the carbon particles with higher surface porosity was explained by the high porous structure that offered a closer proximity to the reactive species and improved diffusion of ethanol and oxygen within the enzyme films. Efficiency of immobilization of NAD + cofactor has been also demonstrated. We have shown the effect of multienzymatic reaction at the anode on the storage and operational stability. A high open circuit potential has been recorded 1.02V, demonstrating the benefit of NAD + immobilization with maximal power density of 300 µW.cm-2. Chronoamperometric measurements show a current density of 90 µA.cm-2 during 20 hours demonstrating the operational stability of the ethanol biofuel cell based on NAD + cofactor immobilization.