Abstract: To address the problem of deep denitrification in low-pollution water, this study investigated the sulfur autotrophic denitrification enrichment process under various sulfur-to-nitrogen ratios (S/N) and hydraulic retention times (HRT). The denitrification performance, sulfur metabolism characteristics, and microbial community succession patterns were systematically evaluated. The results showed that under an HRT of 24 h, the sulfur-nitrogen metabolic coupling was stable, and the correlation coefficient between sulfate production and nitrate removal ranging from 0.949 to 0.977. At S/N = 2:1, it was conducive to the rapid start-up of the reactor, and at S/N = 1:1, it was conducive to the construction of denitrifying bacterial communities. When the operation strategy was to completely remove nitrate and then enter the next HRT, the rapid enrichment of denitrifying bacterial communities could be achieved. However, at S/N = 2:1, the disproportionation of thioglycolate produced S^2-, which inhibited denitrification. Through batch experiments, the denitrification effect was optimal when S/N = 2:1 and HRT = 24h, with a nitrate removal efficiency of 82.83% and a maximum denitrification rate of 1.807 mg N·g^-1 VSS·h^-1. Microbial analysis revealed that Thiobacillus was the dominant autotrophic denitrifying bacterial genus (relative abundance 40.80% - 62.32%); at S/N = 2:1 and HRT = 24h, it was conducive to the screening of Thiobacillus autotrophic denitrifying functional strains, the establishment of sulfur cycle functions such as dark oxidation of sulfide and oxidation of sulfide, and the establishment of three-level pathways such as environmental perception and adaptation. These findings elucidate the synergistic regulatory mechanisms of the sulfur autotrophic denitrification enrichment process under different S/N and operation strategies.