Objective To analyze the relationship between virulence genes and traditional Kanagawa phenomena,based on the virulence genes carried by Vibrio parahaemolyticus isolates from different sources of serovar O3,to provide basic data for further studies on the pathogenicity of Vibrio parahaemolyticus. Methods Multiplex polymerase chain reaction(PCR)and Kanagawa phenomenon assay were performed to detect the existence of virulence associated genes tdh and trh as well as haematolysis of Vibrio parahaemolyticus serovar O3 strains with different origin. The correlation between tdh and trh genes and Kanagawa phenomenon were verified based on test. Results Among 183 Vibrio parahaemolyticus serovar O3∶K6 strains, tdh gene was detected in 182 strains and trh gene exists in the remaining strain, detection rate of the two genes were 99.45% (182/183) and 0.55% (1/183), respectively. The positive rate of Kanagawa phenomenon was 100.00% (183/183). Among 57 Vibrio parahaemolyticus serovar O3 non-K6 strains, detection rate of tdh gene was 3.51% (2/57), trh gene was not detected. Furthermore, the positive rate of Kanagawa phenomenon was 10.53% (6/57). Significant correlation between tdh gene and Kanagawa phenomenon was verified (χ²=1.78, P>0.05), meanwhile, similar between trh gene and Kanagawa phenomenon was not detected (χ²=186.01, P<0.05). Conclusion Significant correlation between tdh gene and Kanagawa phenomenon was verified in Vibrio parahaemolyticus serovar O3∶K6 strains, and pathogenicity of Vibrio parahaemolyticus can be detected by using PCR method rapidly.