Abstract：A double-chamber microbial fuel cell (MFC) was successfully started up by using the supernatant of anaerobically fermented excess sludge as substrate in the anode chamber and potassium ferricyanide as electron acceptor in the cathode chamber. The concentration changes of various volatile fatty acids (VFAs) in the supernatant were investigated during the anaerobic fermentation process of the sludge. Then the electricity production process of the MFC was studied, and the substrate consumption and electron donors during electricity production were also analyzed. The results indicated that the acetic acid concentration was the highest (approximately accounting for 50% of total VFAs), followed by isovaleric acid and propionic acid (respectively about 18% and 15% of total VFAs), n-butyric acid and isobutyric acid (all less than 10% of total VFAs), while the n-valeric concentration was the lowest (less than 1% of total VFAs) among the VFAs in the supernatant of anaerobically fermented sludge. The MFC kept a stable voltage output (0.65±0.05V) for 250h, and the coulombic efficiency was 9.09%. An overall degradation trend was found among the total chemical oxygen demand (TCOD), the soluble chemical oxygen demand (SCOD) and VFAs during electricity production of the MFC. The removal rates of TCOD and SCOD were 74.9%、86.4%, respectively. The complete consumption of VFAs was accompanied by the rapid deterioration of the electricity production performance, indicating that the main electron donors were VFAs. The consumption and production of VFAs existed simultaneously during the process of electricity generation, while the consumption rates were faster than the production rates in general. The consumption rate sequence of various VFAs was as following: acetic acid> n-butyric acid> propionic acid> n-valeric acid> isovaleric acid> isobutyric acid.