Abstract: Fe₃O₄ nanoparticles was made by coprecipitation, and characterized for scanning electron microscope (SEM). The effect of different concentration of Fe₃O₄ nanoparticles on volume gas production and CH₄ ratio during the anaerobic digestion process at the medium temperature of 35 ℃ was examined. The results showed that Fe₃O₄ nanoparticles of 100 mg/L maintained ammonia at the level of 600-1 200 mg/L, pH at 7-8, and improved TCOD_Cr and SCOD_Cr degradation rates by 8.35% and 9.90%, respectively. This amount of Fe₃O₄ nanoparticles produced maximum concentration of volatile fatty acids (VFAs), which can reach 4 300 mg/L, and strengthened the use of acetic acid. Fe₃O₄ nanoparticles of 100 mg/L improved the biogas production. Compared with the control group, the system increased the gas production by 28.08%, decreased the gas production cycles by two days and increased the methane content by 6%.