Electricity can be generated during the wastewater treatment by microbial fuel cell (MFC). Effect of the anode pretreatment method, anode modification and electronic media in anolyte on electricity generation of a single chamber microbial fuel cell (SCMFC) with an air cathode was investigated by performance measurement of the MFC during one cycle of generating electricity. Results show that the discharge time of MFCs with stainless steel anode pretreated by NH4Cl and HCl is longer than that with anode pretreated by the distilled water and NaOH; the performance of MFC with anode pretreated by NH4Cl is the best; comparison with stainless steel anode and adhere activated carbon, the current of MFC with the stainless steel anode adhere the carbon fiber is the maximum and its Coulombic efficiency is the highest; The electricity generation characteristics of the MFC with the anode adhered carbon fiber and pretreated by NH4Cl and the anolyte added the electronic media is the best. The action of the electronic medium and the NH4Cl on the MFC is synergism. It's of great significance that the research results improve the performance of MFC by anode pretreatment.