In this study we report a new simple process to manufacture a biofuel cell consisting of a glucose oxidase (GOx) based anode and a laccase (LAc) based cathode. The process is based on the electro-deposition of the enzymes, conducting polymer and redox mediators from ultrapure water at a potential of 4 V vs. AgCl/Ag. Contrary to the conventional electro-deposition from high ionic strength (buffer solution) at low applied potential (1 V vs. AgCl/Ag) where only thin films could be deposited, leading to BFC with moderate power, the electro-deposition from ultrapure water at 4 V allows the growth of thick films leading to BFC with high power output. It was observed that the combination of polypyrrole (PPy), with ferrocenium hexafluorophosphate (FHFP) and pyrroloquinoline quinone (PQQ) to be appropriate for the electron transfer at the GOx bioanode, while the combination of polypyrrole with bis-(bipyridine)-(5-amino-phenanthroline) ruthenium bis (hexafluorophosphate)(RuPy) and 4,4-sulfonyldiphenol (SDP) to be effective for the electron transfer at the LAc biocathode. The working biofuel cell was studied at 37 °C in phosphate buffer solution at pH 7.4 containing 10 mM glucose and in human serum. Under these conditions, the maximum power density reached 3.1 μW mm −2 at a cell voltage of 0.28 V in buffer solution and 1.6 μW mm −2 at a cell voltage of 0.21 V in human serum. This study offers a new route to the development of enzymatic BFCs with high performance and provides information on enzymatic BFCs as in vivo power sources.