Abstract: To explore the effect of biochar on the advanced purification of tailwater of sewage treatment plant by sulfur-based autotrophic denitrification constructed wetland (SADCW) and the potential microbial mechanism, two vertical subsurface flow constructed wetlands were established, which were filled with sulfur + limestone (CW-C) and sulfur + limestone + almond shell biochar (CW-B), respectively. The concentrations of COD, TN, \mathrmNO_3^- -N, \mathrmNO_2^- -N, \mathrmNH_4^+ -N, \mathrmSO_4^2- , and dissolved oxygen (DO) in effluent at different hydraulic retention times (HRTs) were measured. High throughput sequencing technology was used to analyze the microbial community structure, and the relative abundance of nitrogen and sulfur cycling functional genes was predicted by using the PICRUSt software. Results showed that the average TN removal rates of CW-C and CW-B at HRT=4 h were 6.18%-21.14% and 5.63%-15.92% higher than those of other HRTs, respectively; moreover, the \mathrmNO_3^- -N removal rate of CW-B (mean value 96.66%) was significantly higher than that of CW-C (95.50%, P<0.01) at such HRT. A much lower DO level was created by biochar in the constructed wetland, which increased the relative abundance of the main phyla of sulfur-based autotrophic denitrifying microorganisms, Bacteroidota and Chloroflexi, as well as the main genera, Chlorobium, Geothrix, norank_f_PHOS-HE36, and Thiothrix. At the same time, it also increased the relative abundance of the main phyla of heterotrophic denitrifying microorganisms, Actinobacteriota and Firmicutes, as well as the main genera, norank_f_Caldilineaceae, unclassified_f_Rhodocyclaceae, and Denitratisoma. In addition, the relative abundances of the functional genes (sat, cysH, and sir) related to the assimilatory sulfate reduction process were enhanced in CW-B. The proportion of heterotrophic denitrification was strengthened by biochar addition in SADCW, which was more conducive to \mathrmNO_3^- -N removal. Furthermore, biochar also promoted the assimilatory sulfate reduction process and reduced by-product \mathrmSO_4^2- production. The study clarified the role of biochar in SADCW for the advanced purification of tailwater of sewage treatment plant and could provide a theoretical basis for the regeneration and recycling of water resources.