Recirculating aquaculture systems (RAS) using biofloc biofilters can address the high accumulation of nitrate (NO3 −-N) and total suspended solids (TSS) in in-situ biofloc technology (BFT) aquaculture systems. A RAS was divided into the following four zones: reaction zones of the BFT biofilters, settling zones of the BFT biofilters, culture tanks, and a buffer tank. During the 95-day experimental period, the water quality parameters and microbial communities in different zones of the RAS were compared. The NO3−-N, nitrite (NO2−-N), total ammonia nitrogen (TAN), and TSS in the culture tanks were continuously controlled at average concentrations of 9.84 ± 5.15, 0.28 ± 0.15, 0.10 ± 0.18, and 96.92 ± 33.82 mg·L−1, respectively. Because denitrification produced alkalinity in the BFT biofilters, the alkalinity of the culture tanks reached an appropriate concentration of 109.40 ± 10.57 mg CaCO3·L−1 without intentional adjustment. The concentrations of NO3−-N, NO2−-N and TAN in the reaction zones were significantly lower than those in the influent (buffer tank) and culture tanks. However, there was no significant difference found for major water quality parameters between the reaction zones and the settling zones. There was also some removal of soluble reactive phosphate by the BFT biofilters, although there was no significant difference between the biofilters and influent. Microbial communities in the reaction zones were more diverse than those in the culture tanks, buffer tank and settling zones. Denitrifiers, nitrifying bacteria and important functional microbial populations for phosphorus removal were all present in the reaction zones, and at higher levels than those in other zones. Nitrogen and phosphorus removal were mainly achieved in the reaction zones of the BFT biofilters, and the microbial communities in the other three zones were similar. Besides, Scortum barcoo grew from an individual weight of 347.7± 73.0 to 472.1 ± 106.2 g, achieving a final stocking density of 32.81 kg·m−3 at the end of the experiment.