In order to investigate the pathogen of the diseased tongue sole Cynoglossus semilaevis, three strains of Gram-negative bacteria isolated from diseased C. semilaevis were tentatively named strains ST-1, ST-3 and ST-6, which were identified by physiological and biochemical characteristics, antimicrobial susceptibility test, 16S rDNA gene sequences analysis and histopathology examination. Furthermore, the SYBR Green Ⅰ quantitative real-time PCR (qRT-PCR) targeting the outer membrance protein (OMP) gene was established. The biochemical results of the isolate ST-1, ST-3 and ST-6 were consistent with the properties of V. vulnificus. The BLAST alignments of the 16S rDNA gene sequence showed that the isolates ST-1, ST-3 and ST-6 shared the identities above 99% with type strain Vibrio vulnificus ATCC 29307 (X74727). The phylogenetic tree indicated the isolates clustered with V. vulnificus ATCC 29307. The isolates were eventually identified as V. vulnificus. Antimicrobial susceptibility testing showed that the isolates were highly susceptible to cefixime, cefoperazone, streptomycin, imipenem and florfenicol. The challenge experiments showed that the isolated strains were pathogenic on C. semilaevis and zebrafish(Danio rerio), and similar symptoms were observed with the natural infection. Histopathology examination revealed the hyperplasia of the epithelial cells in the primary lamella, the swelling of the secondary lamella and the increase of the number of granulosa cells and mucus cells, hepatic sinusoid and the congestion of central vein in the liver, hemorrhagic-necrotic lesion in the kidney, the atrophy of the lamina propria and the increase of the number of mucus cells in the intestine. The SYBR Green I quantitative PCR (qPCR) method based on the OMP gene was further established and the results showed that the correlation coefficient of standard curve was 0.995 and the lowest limit of detection was 1.88×10² CFU/mL. The results of this study will lay a foundation for the pathogenesis and prevention of C. semilaevis vibriosis, which can provide scientific reference for the molecular diagnosis of the disease.