Enzymatic biofuel cells (EBFC) are promising sources of green energy owing to the benefits of using renewable biofuels, eco-friendly biocatalysts, and moderate operating conditions. In this study, a simple and effective EBFC was presented using an enzymatic composite material-based anode and a nonenzymatic bimetallic nanoparticle-based cathode, respectively. The anode was constructed from a glassy carbon electrode (GCE) modified with a multi-walled carbon nanotube (MWCNT) and ferrocene (Fc) as a conductive layer coupled with the enzyme glucose oxidase (GOx) as a sensitive detection layer for glucose. A chitosan layer was also applied to the electrode as a protective layer to complete the composite anode. On the other hand, the cathode consisted of a disposable pencil graphite electrode (PGE) modified with platinum-palladium bimetallic nanoparticles (Nps) which exhibit excellent conductivity and electron transfer rate for the oxygen reduction reaction. The EBFC consisting of MWCNT-Fc-GOx/GCE anode and Pt-PdNps/PGE cathode exhibits an open circuit potential of 285 mV and a maximum power density of 32.25 µW cm− 2 under optimized conditions. The results show that the proposed EBFC is a promising candidate for detecting glucose while harvesting power from artificial serum samples.