Abstract: The widespread application of mainstream anaerobic ammonium oxidation (ANAMMOX) technology is limited by the challenge of rapidly enriching high-purity anaerobic ammonium-oxidizing bacteria (AnAOB). Traditional biofilm systems exhibit excellent microbial retention capacity, and AnAOB tend to grow in attached forms. However, due to the slow growth rate of AnAOB and competition for nitrite by denitrifying bacteria (DB), the ANAMMOX biofilm startup period is prolonged, and their abuandance is low, hindering practical implementation. To address this, we propose a strategy of “suspended sludge-assisted startup followed by phased elimination for enrichment” combining the advantages of biofilm systems. In the early stage of system startup, high-concentration suspended sludge is used to provide sufficient species flock and EPS to promote biofilm formation. After system startup, the startup period of biofilm is shortened by regulating the biomass of suspended sludge to promote AnAOB enrichment. A sequencing batch reactor (SBR) was inoculated with pure suspended sludge and equipped with sponge and K3 biofilm carriers. After 70 days of startup cultivation, a stepwise discharge of suspended sludge was implemented (partial discharge on day 71 and complete removal on day 141). By day 210 of continuous operation, a stable pure biofilm system was successfully established. Results demonstrated that this strategy strengthened the dominant role of ANAMMOX in nitrogen removal, increasing its contribution from 83.53% to 91.34%, while the total nitrogen removal efficiency (NRE) improved from 81.94% to 88.55%. 16S rRNA sequencing revealed that the dominant AnAOB genus in the biofilm was Candidatus_Jettenia, whose relative abundance increased by 5.13% as floccular sludge was gradually discharged. Metagenomic analysis further confirmed that Candidatus_Jettenia dominated the expression of key nitrogen metabolism enzymes genes in the biofilm. After management, the abundance of NarG genes increased, while the abundance of NorB genes decreased, promoting the conversion of NO₃⁻ to NO₂⁻, accelerating the accumulation of ANAMMOX reaction substrates, and thereby enhancing the nitrogen cycling process driven primarily by ANAMMOX. Thus, the dynamic regulation strategy of "suspended sludge-assisted startup followed by phased elimination for enrichment" provides a simple yet effective method for efficient AnAOB enrichment in biofilms, offering promising potential for scaling up mainstream ANAMMOX technology in real wastewater treatment applications.