In China, aquaculture accounts for over 80% of aquatic product supply, playing a vital role in safeguarding national nutrition and driving economic growth. However, the substantial annual discharge of aquaculture wastewater has raised serious environmental-related concerns. As a result, the removal of nitrogen and phosphorus from such wastewater has become essential for promoting the sustainable development of the aquaculture. Although many multi-stage treatment systems, often referred to as “three ponds and two dams”, have been constructed to treat aquaculture tailwater, key operational parameters, such as hydraulic retention time (HRT) still lack scientific justification. Therefore, our study established a multi-stage constructed wetland system (MCWS) consisting of a sedimentation pond (SP), filtration dam A (FDA), an aerobic pond (AP), filtration dam B (FDB), and an ecological purification pond (EP). We investigated the effect of HRT on nitrogen and phosphorus removal and explored the associated microbial mechanisms. The system was operated continuously for 142 days under four HRT conditions (24 h, 48 h, 96 h, and 192 h), during which we monitored pollutant removal efficiencies and microbial community dynamics. The results showed that with the extension of HRT, the removal efficiency of TN and TP gradually increased. When the HTR was 192 hours, TN decreased from 25.74mg/L to 2.59mg/L, TP decreased from 5.78mg/L to 0.92mg/L, and the removal rates were 89.77% and 80.91%, respectively. Functional units exhibited clear synergistic effects: denitrifying bacteria in SP facilitated heterotrophic nitrate reduction; photosynthetic nitrogen-fixing bacteria and polyphosphate-accumulating organisms thrived in the high-dissolved-oxygen environment of AP; and cyanobacteria in EP contributed to photosynthetic nitrogen fixation coupled with nitrate assimilation, enabling advanced nitrogen removal. This study clarifies the microbial metabolic pathways and pollutant removal mechanisms within each unit of the MCWS, providing a theoretical basis for the engineering design and operational optimization of multi-stage treatment systems for aquaculture wastewater.