In order to determine the electrochemical activity of Shewanella decolorationis S12, anaerobic grown cells were scanned by the cyclic voltammograms (CV). The results of CV showed that strain S12 displayed certain electrochemical activities and could be used as a catalyzer working in microbial fuel cell (MFC) to produce electricity. The effects of different electron donor, such as lactate, formate and pyruvate with different concentration on the production of electricity were analyzed by using strain S12 in a single chamber MFC with a PEM/electrode assembly sandwiched type. The results showed that lactate was the optimal electron donor and the concentration of the electron donor determined the electricity generation capacity and potential development in the MFC. When 10 mmol/L and 20 mmol/L of lactate were used, the maximum power density of 21.93 mW/m2 and 55.72 mW/m2 were obtained, respectively.