［Purposes］ In order to solve the technical bottleneck problem of practical engineering applications where the presence of organic matter affects the application of single-stage autotrophic nitrogen removal system, and promotes the solution of the water pollution problem in Dongting Lake, the effect of different carbon to nitrogen ratio (C/N) values on the nitrogen removal performance of the single-stage autotrophic nitrogen removal system was investigated, and the best C/N value that the process can withstand was explored. ［Methods］ Taking the self-innovation air-lift internal circulation reactor as the research object, the method of long-term continuous experiment combined with ectopic activity batch experiment was adopted. By adjusting and controlling important process parameters such as reactor aeration, temperature, pH, influent ammonia nitrogen and organic matter concentration, collecting sludge samples in the reactor periodically, determining sludge concentration based on chemical analysis and instrumental analysis, and using high-throughput sequencing technology, we investigated the effects of different C/N values on the long-term continuous operation performance of the process and the microbial community succession. ［Findings］ (1) Through long-term continuous operation monitoring for 364 days, the total nitrogen removal efficiency of the reactor can be maintained above 80% when the influent C/N value is from 0.0 to 2.0, and the highest nitrogen removal performance was achieved when the C/N was 2.0. When the C/N of the treated wastewater exceeds 2.0, the nitrogen removal performance of the reactor decreases, and the operation time to reach stability is longer. (2) The removal rate of organics can be maintained at a high level when each operation stage of the process is stable. (3) The dominant genus of anammox bacteria in the reactor was Candidatus Brocadia, the dominant genus of ammonia-oxidizing bacteria was Nitrosomonas, and the dominant genus of denitrifying bacteria was Denitratisoma. The relative abundance of Candidatus Brocadia kept decreasing with the increase of C/N ratio, and the relative abundance of Nitrosomonas kept decreasing with the increase of C/N ratio. ［Conclusions］ Considering both the operational performance and the economic cost, the optimal C/N ratio for the air-lift internal circulation and single-stage autotrophic denitrification process was 1.0. With the optimal C/N ratio, the abundance and activity of ammonia-oxidizing bacteria and anaerobic ammonia-oxidizing bacteria were not the strongest, but the synergistic effect of the functional groups in the process maintained a high level of denitrification performance. The nitrogen removal performance decreased after the C/N value exceeded the optimum value.