Biofuel cells are bio-electrochemical devices, which are suitable for environmentally friendly generation of energy. Enzymatic biofuel cell (E-BFC) operating at ambient temperature and in low fuel concentrations is reported in this research. Glucose oxidase (GOx) from Penicillium funiculosum 46.1 and insoluble redox mediator 9,10-phenantroline-5,6-dione (PD) immobilized on graphite rod electrode (GRE) were applied in the design of biofuel cell anode (GOx/PD/GRE). Horseradish peroxidase (HRP) modified GRE was applied in cathode (HRP/GRE) of the same biofuel cell. GOx/PD/GRE anode demonstrated sufficient electro-catalytic activity during the oxidation of glucose, and HRP/GRE cathode exhibited direct electron transfer based electro-catalytic reduction of hydrogen peroxide. Both anode and cathode were operating in buffer, pH 6.0, consisting of 0.05 mol L-1 sodium acetate-sodium phosphate and 0.1 mol L-1 KCI. The maximal registered open-circuit-voltage of the complete E-BFC exceeded 640 mV, calculated maximal power density was 4.2 mu W cm(-2) at the cell voltage of 530 mV in the presence of 200 mmol L-1 or higher glucose and 0.1-0.5 mmol L-1 of hydrogen peroxide concentration in anode and cathode compartment, respectively. Such new design of biofuel cell allows to utilize glucose as a fuel for the anode, and H2O2 formed during the catalytic action of GOx, which is immobilized on anode, could be utilized as a fuel at the cathode of the same biofuel cell. (C) 2014 Elsevier B.V. All rights reserved.