<p>Microbial fuel cell (MFC) is a bio-electrochemical technology which can convert organic compounds into electrical energy through microorganisms. The types of carbon source in anode and electron acceptor in cathode are considered the main factors influencing the stability and efficiency of dual-chamber MFC. In the current study, five substrates including acetate, lactate, glucose, sucrose, and humic acid were chosen to investigate the effect of carbon source on the performance of MFC using ferricyanide as an electron acceptor. The electrical performance of the MFC was closely related to the substrate type. The ferricyanide cathode MFC fed with acetate had the lowest internal resistance (109 Ω) and the highest power density 1277 mW∙m⁻². The substrate with lower molecular weight exhibited better electrical generation and more stable operation. This can be attributed to complete formation of biofilm on the anode surface using acetate or lactate as a carbon source. The effect of electron acceptors was also evaluated by replacing ferricyanide with air oxygen. Compared to oxygen cathode, the ferricyanide cathode MFCs were more suitable for lab-scale experiment due to their lower internal resistances and greater power densities. COD removal efficiencies of the MFCs using ferricyanide and oxygen were similar. The results indicated that acetate and ferricyanide were the best combination for MFC start-up and long-period operation. High molecular weight substrates such as glucose, sucrose, and humic acid were not suitable as the sole carbon source for MFC.</p>