In order to determine the pathogen, which can cause the death of cultured Paralichthys olivaceus from ascites disease in Beidaihe area, Hebei Province, three dominant bacteria were isolated from P. olivaceus infected with ascites. The biological status of the isolates was determined by physiological and biochemical identification and 16S rRNA sequence alignment. The pathological characteristics of the isolates were further identified by virulence genes (toxR, vhhA, vhhB) and histopathological analysis. The results showed that the three isolates were Vibrio harveyi and the virulence genes of the three isolates were all positive. Pathological sections showed that the isolates could be used to damage multiple organs (intestine, kidney, spleen and liver) of P. olivaceus. The LD₅₀ of strain BDHYPFS-Y1G to P. olivaceus was 5.88×10⁶ CFU/g, which was lower than the toxicity of natural state. Drug susceptibility test indicated that all three isolates were highly sensitive to the nitrofurantoin. This study confirmed the pathogen of this ascites disease, and preliminarily studied the pathogenicity and drug sensitivity of the pathogen, which can provide scientific basis for the prevention and control of the disease in industrial culture of flounder.