An enzymatic biofuel cell was prepared based on covalently linked glucose oxidase (GOx) and bilirubin oxidase (BOx) as the anodic and cathodic catalyst, respectively. We performed the covalent co-immobilization of GOx and a ferrocene derivative 1,1'-ferrocenedicarboxylic acid, as an electron transfer mediator, on a graphite carbon felt electrode. In an effort to explore a new type of bioanode, each step of the electrode preparation was examined by infrared spectroscopy, electrochemical impedance spectroscopy, and cyclic voltammetric study. The biofuel cell parameters were attained in a neutral buffer solution containing 20 mM of oxygen-saturated glucose at ambient temperature. The open-circuit voltage reached 0.49 V. and the maximum power density generated from the biofuel cell was 13 mu W cm(-2) at a cell voltage of 0.25 V. The results show that the approach proposed in this study allows for the electron transfer between the enzyme and electrode to be effectively mediated through the co-immobilized mediator and that it should be considered a promising method for the fabrication of enzymatic biofuel cells. (C) 2011 Elsevier B.V. All rights reserved.