Abstract: The effects of both the concentration of corn stalk hydrolysis solution and the volume of activated sludge as an anode substrate on the performance of the double chamber microbial fuel cells (MFCs) were investigated. The double chamber MFCs were built with K₃Fe(CN)₆ as the catholyte. The results show that with the increase in the activated sludge volume from 1.5 to 6.0 mL, the electricity generation of MFCs increases gradually, but it decreases when the activated sludge volume reaches 7.5 mL. As the mass concentration of corn stalk hydrolysate is 0, 10, 15, 20, 30, 40 g/L, the stable voltage of MFCs is 54, 157, 248, 208, 170 and 146 mV, respectively. The best performance of MFCs is obtained with the power density of 54.6 mW/m² and the internal resistance of 496 Ω as the activated sludge volume is 6 mL and the corn straw hydrolysate is 15 g/L. Moreover, the cyclic voltammetry curve (C-V) and electrochemical impedance spectroscopy (EIS) tests prove that the electrode process is controlled by both the charge transfer and the diffusion process, while the reaction process is controlled by the electron transfer.