Abstract: Electroactive biofilms were formed on carbon paper under constant external resistance of 1 000 Ω using Escherichia coli as inoculum and glucose as substrate. In this paper, the performance of the biofilm growth of the microorganism directly on the anode was studied. To form a mature biofilm, five fed-batch cycles were repeated with every period of 1 day. The electrochemical characterization of the microbial fuel cell was evaluated using cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and polarization behavior. It was evident that the enhanced MFC performance was associated with the development of the biofilm. With formation of the mature anode biofilm, the anode polarization resistance was decreased by 66.5%, the anode potential also gradually decreased, while peak output power density was enhanced over 260%.