Abstract: The purpose of this study was to identify the pathogen of white spot disease of Hapalogenys nitens and analyze its antibiotic sensitivity, which could provide scientific basis for diagnosis and prevention of the disease. The pathogenic microorganisms of H. nitens with typical symptoms were isolated by plate separation method. Pathogenic bacteria are identified with morphological, physiological and biochemical characteristics and molecular sequencing techniques. Furthermore, the drug sensitivity test was carried out on the isolated pathogenic bacteria. The pathological section showed that white inflammatory granuloma lesions were observed in the liver, spleen, gills, head kidney and heart of the fish infected. Single colony named hn-1 can be isolated from the spleen, liver and head kidney of the infected and identification of physiological and biochemical characteristics, gene sequencing and regression infection experiments confirmed that Vibrio harveyi was the pathogenic bacterium of visceral white spot disease of the fish infected. Under the electron microscope, the isolated bacterium was observed to have polar flagella and lateral flagella. The virulence gene test showed that the isolates carried a variety of virulence genes, including toxR, luxR, vvh, vhh and trh. Results of drug sensitivity test showed that the isolated strains were mainly sensitive to quinolones, aminoglycosides, tetracyclines and other drugs. The results can provide scientific basis for the prevention and control of visceral white spot disease of H. nitens.