Achieving anaerobic ammonium oxidation (Anammox) in low ammonia nitrogen wastewater represents a significant challenge for municipal wastewater treatment plants. This study employed a mainstream Anammox continuous-flow oxygen-limited biofilm system (DO: 0.4~0.7mg/L) and investigated the nitrogen removal performance for treating simulated domestic sewage with low ammonia nitrogen content under different influent carbon-to-nitrogen (C/N) ratios (C/N: 2~5). Long-term experimental results indicated that a C/N ratio of 3.5~4.5 achieved over 85% total nitrogen (TN) removal efficiency. There was a positive correlation between activity of Anammox bacteria (SAA) and the nitrogen removal efficiency, with the highest SAA observed at a C/N ratio of 4. Additionally, the C/N ratio significantly influenced the extracellular polymeric substances (EPS) composition of the biofilm, with the highest protein-to-polysaccharide ratio (PN/PS) and most stable biofilm structure observed at C/N:4. Metagenomic analysis identified Candidatus Kuenenia stuttgartiensis and Candidatus Brocadia sinica as the dominant species, with relative abundances of 21.5% and 4.7%, respectively. A diverse microbial community, including complete ammonia oxidizers (Comammox), and heterotrophic denitrifiers, contributed to microbial community structure in the system. Nitrogen metabolic analysis further uncovered the involvement of partial denitrification genes (HZS、hdh) and Anammox genes (HZS、hdh) in nitrogen removal processes, thus ensuring stable and efficient mainstream nitrogen removal. Research findings are expected to provide a novel option of mainstream anammox-based nitrogen removal process for wastewater treatment plants.