This study investigates the influence of initial pH on anodic biofilm formation of microbial electrolysis cells. Initial pH affects the activities of microorganisms in MEC, and furthermore will affect hydrogen (H₂) generation. Therefore, we explore the effects of initial pH on anodic biofilm formation of microbial electrolysis cells and the intrinsic reasons. In a single-chamber MEC, with different initial pH (which are 5.0, 6.0, 7.0, 8.0, and 9.0), the maximum power density during the enrichment process was 2.73 mA/cm² at pH 8.0, which is 56% and 23% higher than those working at pH 7.0 and 9.0, and get worse under acidic conditions. pH 8.0 also showed the highest coulombic efficiency of 46.4% compared with other experimental groups, and energy recovery efficiency is 17.5%. Membrane biomass, as an indicator for anode microbial biomass, decreased sharply at pH 5.0 and 6.0 compared with the neutral and alkaline. Scanning electron microscopy verifies that alkalescent conditions are beneficial to form more rod-shaped bacteria in MECs. These results show that electrochemical interactions between bacteria and electrodes in MECs are enhanced under neutral and alkaline conditions, and the optimal initial pH for anode bacteria formation of 8.0. The information provided below will be useful for improving MEC hydrogen generation.