Macrobrachium rosenbergii is one of the most economically important freshwater shimp, with fast growth and high nutrient content in the agricultural development of China. However, it had been suffering diseases infection, causing mass death and great economic losses. In the present study, a bacteria strain was isolated from the diseased zoea of M. rosenbergii and was identified as Vibrio vulnificus by biochemical characteristics and 16S rRNA homologous analysis. The infection test showed that the strain GXFL1-3 was pathogenic to zoea and postlarva of M. rosenbergii, and the half lethal dose (LD50) were 1.16 × 106 CFU/mL and 1.45 × 106 CFU/mL, respectively. Detection of virulence-associated genes by PCR indicated that GXFL1-3 was positive for fur, OmpU, acfA, flaA, vvhA, vvp and tcp, the detection of extracellular enzymes and hemolysin showed that GXFL1-3 was positive for protease, amylase, lecithin, urease and hemolysin activity, further supporting its pathogenicity. A duplex PCR for rapid detection of V. vulnificus was established. Only V. vulnificus could amplify two specific bands of flaA and fur, while the other six strains of Vibrio were negative. The minimum detectable amount of template was 2.4 × 103 CFU/mL through sensitivity test.