In order to improve the operation performance of microbial fuel cells, improved the degradation rate of nitrate and the power output of microbial fuel cell, a typical single chamber air-cathode microbial fuel cell (AC-MFC) is inoculated and operated with urban sewage treatment plant clarifier sludge as inoculum source and sodium nitrate as electron acceptor. It is successfully started by synthetic wastewater containing a phosphate buffered nutrient solution (PBS, 50 mmol/L) and sodium acetate (1 g/L). After successful starting, the four factors of carbon source, C/N, nitrate concentration and temperature are considered to optimize the operation performance of MFC. The test result shows that the operation performance of MFC is best under the conditions of anhydrous sodium acetate as carbon source, C/N of 5∶1, 200 mg/L nitrate concentration and at 30 ℃, and the degradation rate of nitrate reaches more than 90% and the voltage of MFC is 0.462 V. After 6 cycles of operation, the voltage and power density of MFC reaches 0.62 V and 4.53 W /m2. AC impedance analysis indicates that the MFC resistance is 130 Ω. Scanning electron microscopy of electrode surface illustrates that the number of microbial species are significantly increased. The results indicate that MFC can be an effective technology for nitrate contained wastewater treatment and energy production.