In the present report we focused on the substitution of metallic catalysts by biocatalysts to develop a high efficient biofuel cell. A bioanode and a biocathode were designed using ADH and laccase, respectively. Carboxylated multiwall carbon nanotubes (HOOC-MWCNTs) and polydiallyldimethylammonium chloride (PDDA) were used for immobilizing the enzymes on either polymethylene green (PMG) modified glassy carbon or graphite electrodes. In this way, an ethanol–oxygen biofuel cell was designed in which PDDA/ADH/PDDA/HOOC-MWCNTs/PMG/GC and PDDA/Lac/PDDA/HOOC-MWCNTs/PMG/Gr operated as bioanode and biocathode, respectively. In the optimized condition of O2 saturated PBS (0.1M, pH 7.5) containing 1mM ethanol and 1mM NAD+ the open-circuit voltage reached to a plateau at 504mV based of which the power density of 3.98mWcm−2 was obtained.