Abstract: The proper concentration of metal ions in anode of microbial fuel cells (MFCs) could promote the growth of microbes and increase the power generation ability. In order to understand the effect of Cu²⁺ in anode of MFC, the effects of Cu²⁺ concentration in anode on MFC operation, the removal capacity of Cu²⁺, and the migration and transformation of Cu²⁺ were studied. The species and valence states of the material produced on the surface of the microorganism in anode of MFCs were also systematically analyzed. The migration and transformation of Cu²⁺ and its effect on electricity generation performance were investigated in anode of MFCs. The results showed that the maximum power density increased to 536.6 mW/m² by adding Cu²⁺ (5.54-88.64 mg/L) in anode of MFC. The removal rate of Cu²⁺ was more than 95% by adding Cu²⁺ (5.54-88.64 mg/L) in anode of MFCs. Most of the Cu²⁺ (89.24%) was adsorbed or reduced by biofilm, while 6.15% Cu²⁺ was deposited in the anode chamber bottom, 3.12% Cu²⁺ was attached on the surface of the graphite anode, and only a very small amount of Cu²⁺ (less than 0.1%) was migrated to the cathode. The minimum toxic concentration (MTC) of Cu²⁺ for MFCs was 22.16 mg/L. The scanning electron microscope-energy dispersive spectrometer (SEM-EDS), X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS) analysis showed that most of Cu²⁺ (63.56%) was reduced to Cu⁺ and Cu⁰ on the surface of biofilm in anode of MFCs. This discovery will provide a new concept for the removal and recovery of heavy metals from organic wastewater by MFCs.