Abstract: Eight pilot-scale vertical flow constructed wetlands (VFCWs) with different treatments were constructed to purify aquaculture tailwater. The study aimed to assess the effects of adding the antibiotics florfenicol on the removal of organic matter, nitrogen, and phosphorus, as well as to explore mitigation strategies through substrate optimization and planting. The results showed that the removal rates of COD_Cr, TN, and TP ranged from 54.28% to 76.41%, 61.60% to 89.18%, and 55.47% to 87.00%, and the presence of antibiotics decreased the removal rates of COD_Cr, \rmNO^-_3 -N, and TN by 11.72% to 22.21%, 9.23% to 19.44%, and 7.24% to 16.40%, respectively. IVCWs with plant exhibited higher nitrogen and phosphorus removal efficiencies compared to those without plant. Notably, planting significantly enhanced the removal rate of florfenicol by 22.98% to 31.87%. While the addition of biochar did not significant effect the removal of conventional pollutants, it decreased the removal rate of florfenicol. Antibiotic addition, biochar addition, and planting had significant effects on microbial community structure. Proteobacteria was the most dominant bacterial phylum in VFCWs, playing a crucial role in nitrogen and antibiotic removal. The relative abundance of Patescibacteria was higher in IVCWs with florfenicol addition and planting than others, which was significantly and positively correlated with florfenicol removal, and might be the key phylum for the removal of florfenicol. Overall, VFCWs could effectively remove conventional pollutants and antibiotics from aquaculture tailwater, and plants could synergize with microorganisms to mitigate negative effects of antibiotic on the pollutants removal capacity of VFCWs.