The water purification process includes filtration of arc screen, strong aeration, biological filtration and UV sterilization in the recirculating aquaculture system (RAS). The pathogens of skin ulceration, Vibrio splendidus and Vibrio harveyi were selected to be the indicator bacteria for study on bacterial distribution and extermination in RAS of flatfish Cynoglossus semilaevis. Pathogens were found in the feed residues, organic sediment on the tank wall and each instrumental compartment of RAS. Unhealthy fish seedlings and tools used in the cultivation could also bring pathogens into the culture system. Extermination trials illustrated that the filtration of arc screen, strong aeration and UV sterilization were important in the elimination of bacteria. The disinfection rate of pathogens in the organic sediment on tank wall was higher than 90% by washing with KMnO4 at a concentration of 25×10-6 mol/L for 1.5min. While the disinfection rate of pathogens on the body surface of the flatfish reached 94.49% by being soaked in H2O2 at a concentration of 100×10-6 mol/L for 10min. This is the first report on the distribution and extermination of pathogenic bacteria in RAS of flatfish, which will provide theoretical data and reference for the management of RAS.