The pathogen was isolated from lesion and was identified by artificial infection in order to investigate the pathogen of skin ulcer of Cynoglossus semilaevis. The morphological, physiological and biochemical characteristics, and the activity of extracellular products and hemolysin and the the sensitivity to the antimicrobial agents were studied, the 16S rRNA and gyrB genes were partially sequenced and compared with sequences deposited in GenBank, and the molecular phylogenetic trees were constructed. Four strains of dominant bacteria associated with serious skin ulcers were isolated from half-smooth tongue sole, C.semilaevis. Bacterial strain A3 was proved to be a pathogen by muscle injection with bacteria suspension, and the LD50 was 1.5×104.2 CFU/mL. The GenBank accession no·was JN391271 and JN 168881 respectively of sequenced 16S rRNA gene and gyrB gene of isolate. 16S rRNA and gyrB genes exhibited high similarity with Vibrio harveyi from GenBank database. The results of physiological and biochemical tests and molecular identification suggested that the pathogen was V. harveyi. Detection of the activity of extracellular products and hemolysin showed that the strain could produce amylase, lipase, urease, lecithinase, proteinase and with β haemolysis in containing 4% defibrinated rabbit blood of TSA plates, but no gelatinase. The results of drug resistance of the pathogen to 17 antimicrobial agents showed that the strain was sensitive to tetracycline, doxycycline, norfloxacin, sulfafurazole and so on, but lowly sensititve or resistant to other tested antimicrobial agents.