Display omitted •The increasing influent COD could stimulate the production of EPS.•TB-EPS were higher than LB-EPS, benefitting the formation of biofilms.•Carbohydrates, proteins, phenols and alcohols were the main components of EPS.•The abundance variation of Anammox bacteria was similar with the EPS contents.•Candidatus Kuenenia was the dominant genus of Anammox, rising from 3.26% to 12.38%. The biofilm system is beneficial for Anammox process designed to treat landfill leachate. In this study, the composition of extracellular polymeric substances (EPS) and the microbial community in an Anammox biofilm system were analyzed to determine the functions driving the biofilm’s ability to treat landfill leachate. The results demonstrated that increasing influent carbon oxygen demand (COD) could stimulate EPS production. EPS helped enrich Anammox bacteria and supplied them with nutrients and enzymes, facilitating effective nitrogen removal (approximately 95%). The variation in Anammox bacteria was similar to the variation in EPS composition. In the tested Anammox Sequencing Biofilm Batch Reactor (SBBR) system, Candidatus Kuenenia was dominant among known Anammox genus, because of its high substrate affinity and because it adapts better to landfill leachate. The relative abundance of Candidatus Kuenenia in the biofilm rose from 3.26% to 12.38%, illustrating the protection and enrichment offered by the biofilm in carrying out Anammox.