During the survey on diseases of Fugu obscurus from 2002 to 2004, two strains(J-1 and H-3) of pathogenic bacteria with typical symptoms were isolated from abdominal cavity and intestinal mucus of suffering Fugu obscurus. The two isolated strains were identified as non01strains of Vibrio cholerae through biochemical test and serotype identification. It could be inferred that they are pathogens of bacterial enteritis of Fugu obscurus since the results of artificial infection are the same as the natural infection. In order to testify the classification of the isolated strains, the isolated strains were amplified by polymerase chain reaction(PCR) with two primers designed according to the sequence of Vibrio. The PCR products were DNA sequence of 451 bp. The result showed that systematic status between J-1, H-3 and N92001, N16961 was close. Enterotoxin with active subunit A named ctxA is one of the primary infection agents of Vibrio cholerae. While the pathogenicity is related with ctxA, the test of gene of enterotoxin is the most important step of diagnosis. Using N16961 strains as positive control, the same PCR DNA sequence of 400 bp was obtained in the PCR analysis. By sequencing and alignment, this 407 bp sequence of H-3 strains is identical with ctxA, which indicated that J-1 strains and H-3 strains were the pathogenic bacteria of F. obscurus. Bacterial diseases are common and frequent in process of aquaculture especially the intensive aquiculture. This paper shows introduces how to combine common method and PCR to isolate, identify and study bacteria. The pollution of non01strains of V. cholerae in aquatic product reflects in some way the degree of environmental pollution. Thus the study of pathogenicity is meaningful to evaluate if bacteria would cause disease and to limit its prevalence, so as to study and control the zoonosis.