Abstract: The widespread application of anaerobic ammonium oxidation (ANAMMOX) technology is limited by the challenge of rapidly enriching high-purity anaerobic ammonium-oxidizing bacteria (AnAOB), especially under low-nitrogen wastewater conditions. To address this, we propose a strategy of "suspended sludge-assisted startup followed by phased elimination for enrichment" based on biofilm systems. In this strategy, high-concentration suspended sludge was used to provide sufficient inoculum and extracellular polymeric substances to promote biofilm formation during startup, after which the suspended sludge biomass was regulated to reduce substrate competition from denitrifying bacteria, thereby promoting AnAOB enrichment. A sequencing batch reactor was inoculated with pure suspended sludge and equipped with sponge and K3 biofilm carriers. The suspended sludge was partially and completely discharged on days 71 and 141, respectively. After 210 days of continuous operation, a stable pure biofilm system was successfully established. During operation, nitrogen removal performance was monitored, and 16S rRNA sequencing and metagenomic analyses were conducted to investigate microbial community structure and nitrogen metabolism gene dynamics. Results demonstrated that after sludge regulation, the total nitrogen removal efficiency improved from 81.94% to 88.55%, while the nitrogen removal contribution rate of ANAMMOX increased from 83.53% to 91.34%. The dominant AnAOB genus in the biofilm was Candidatus_ Jettenia, and the relative abundance of AnAOB increased by 5.13 percentage points. Meanwhile, the abundance of narG genes increased, while the abundance of norB genes decreased, promoting the conversion of \mathrmNO_3^- to \mathrmNO_2^- , accelerating the accumulation of ANAMMOX reaction substrates, and thereby enhancing the nitrogen cycling process driven primarily by ANAMMOX. This study demonstrates that the "suspended sludge-assisted startup followed by phased elimination for enrichment" strategy effectively promoted the enrichment of AnAOB in biofilms, providing new insights for the practical application of ANAMMOX technology in wastewater treatment.