Abstract: Recirculating aquaculture system (RAS) can make the intensive fish production compatible with the environmental sustainability in fish farming. However, high operating cost and low stability of water treatment have limited its application in recent years. In this study, a sequencing batch double-cycle RAS was constructed, in order to increase the stocking density and survival rate for the better RAS water treatment stability. The special designed RAS was designed and operated from April to October 2019, in Shenzhen city, Guangdong Province, China. Both biofilter and mechanical filter were concurrently operated in the double-cycle RAS. The biofilm reactor was run in a 6 h cycle, where each cycle comprised of feeding (30 min), aerobic reaction (4 h), settling (1 h) and discharge (30 min). Hydraulic retention time increased with the intermittent biofilter, in order to completely degrade nitrogen pollutants. The arc screen was run continuously to remove solid particulate matter timely. A systematic investigation has been conducted to explore the system startup and its effect on high density grouper stocking. Nitrifying biofloc was first precultured as the seed sludge to startup the biofilm reactor. It took about 22 days to cultivate the biofilm on sponge fillers using the inoculation with nitrifying biofloc. The mature biofilm showed the high removal efficiency of nitrogen. In effluent, the concentrations of ammonia nitrogen and nitrite nitrogen approached 0 and 0.1 mg/L, respectively. The sponge fillers with matured biofilm were transferred to the biofilm reactor, and then a grouper culture experiment was carried out with the initial stocking density of 30.03 kg/m3. In 0-28 days of culture, the biofilm on the sponge fillers was first detached as the water flow, followed by the regrowth after acclimation. The removal rates of ammonia nitrogen and nitrite nitrogen decreased as the biofilm was detached, whereas, the ammonia concentration in culture water increased to 4.62 mg/L in day 12. High concentration of ammonia induced the grouper no eating during 9-16 d. However, the grouper was return to normal on 17th day, as the concentration of ammonia was far below the reported safe level. The removal rates of ammonia nitrogen and nitrite nitrogen gradually increased to 93.73% and 93.50%, respectively, as the biofilm regrew on the fillers. The final concentrations of ammonia nitrogen and nitrite nitrogen were stabilized at 0.05 and 0.1 mg/L, respectively. In addition, an aerated arc screen combined with foam separation and arc screen can remove fine particles, where obviously decrease the turbidity of culture water when the particle size was below 100 μm. The turbidity of culture water decreased from 5.3 NTU to 1.00 NTU, while kept the level in the subsequent culture experiment. Finally, the average weight of grouper increased from 273±12.22 g to 552.52±107.04 g, after 66 days culture, while the final stocking density reached 60.78 kg/m3, and the survival rate was 100%. This sequencing batch double-cycle recirculating aquaculture system has demonstrated the highly efficient water treatment, high stocking density, and high survival rate. Therefore, the proposed system can be expected for a wide application in fish farming in the near future.