The core of bioelectrochemical systems (BESs) is electrochemically active microorganisms (EAMs), which exert spatial heterogeneity on electrode surface and influences BESs performance. Setting an optimal potential is an effective strategy for improving and optimizing BESs performance, however, how the electrode potential affects spatial structure of microbial community within anode biofilm is not known. Using a complex substrate-fed BES with a wastewater inoculum, this study investigated the community structure and composition of the stratified biofilm developed under the potential of −0.3 V, 0 V, +0.3 V and +0.6 V (vs. saturated calomel electrode) by freezing microtome method and high-throughput sequencing analysis. The spatial heterogeneity of biofilm community was found to be dependent on the electrode potential and a less-stratified community structure was observed for +0.6 V than other potentials. Within the biofilms, the inner layers selected more Geobacter and the outer layers enriched more Acinetobacter and Serratia, potentially suggested a stratification of electron transfer pathway and metabolite-based interspecies communications. The results demonstrated the response of spatial heterogeneity of anode biofilm community to the change of electrode potential, which help to understand the selective and enrichment of kinetically efficient anodic microbiome by electron potential.