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In a bioelectrochemical system (BES), microbial community of anode biofilm is crucial to BES performance. In this study, the stratified pattern of community structure and activity of an anode‐respiring biofilm in a BES fueled with brewery wastewater was investigated over time. The anode biofilm exhibited a superior performance in the removal of ethanol to that of an open‐circuit system. The electrical current density reached a high level of 0.55mA/cm2 with a Coulombic efficiency of 71.4%, but decreased to 0.18mA/cm2 in the late stage of operation. A mature biofilm developed a more active outer layer covering a less active inner core, although the activities of the outer and inner layers of biofilm were similar in the early stage. More Geobacter spp., typical exoelectrogens, were enriched in the outer layer than in the inner layer of biofilm in the early stage, while more Geobacter spp. were distributed in the inner layer than in the outer layer in the late stage. The inactive and Geobacter‐occupied inner layer of biofilm might be responsible for the decreased electricity generation from wastewater in the late stage of operation. This study provides better understanding of the effect of anode biofilm structure on BES performance.