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An anaerobic exoelectrogen named strain CL-1, related to Geobacter sulfurreducens subsp., was isolated and characterized in this work. The growth conditions ranging from the temperature, pH and salinity tolerance to the utilization of different carbon source, and other relevant physiological and biochemical properties were optimized. Furthermore, for better understanding of the electron transfer mechanism, UV/Vis spectra was utilized by in situ linking electrochemistry at different potentials with a three-electrode system. The results indicate that strain CL-1 is an anaerobic bacterium with the optimum growth temperature at 37 °C, the optimum growth pH of 8.0–8.5, and could survive in medium containing 10 g L−1 NaCl. Strain CL-1 can use acetate and ethanol as substrate to produce current densities of 975 ± 25 μA cm−2 and 735 ± 15 μA cm−2 in three-electrode system, respectively. The thickness of the strain CL-1 biofilm formed on the working electrode was around 30 μm. In addition, it was found that the cytochrome c is related to the current generation process by linking electrochemistry and spectroscopy.