Abstract: Some factors affecting single chamber direct microbial fuel cell performance such as iron concentration in the cathode electrode,substrates in anode chamber,substrate concentration and anode electrode area were studied with a single chamber direct microbial fuel cell.It was proved that the load voltage increased with the Fe³⁺ concentration increasing under the same conditions.Electricity was enhanced as more electrons and protons were released during anode chamber reaction for the different substrates.The load voltage was also grown with the increase of the substrate concentration,but the saturation value of the substrate concentration was 0.72 g·L^-1.The more Rhodoferax ferrireducens absorbed on the surface anode electrodes to transport the more electrons,and the load voltage was not multiple of the number of anode electrode.This study provided scientific base for the application of the single chamber direct microbial fuel cell.