Vibrio alginolyticus, a Gram-negative bacterium, is a dominant bacterium in the marine environment. At the same time, it is also a common pathogenic bacterium in daily life. When the temperature is between 25 ℃ and 35 ℃, the epidemic vibriosis is particularly easy to break out, which has a significant impact on the mariculture industry. RseB gene was successfully deleted with Overlap PCR and homologous gene recombination. The results showed that the RseB gene was correlated with the growth rate of V. alginolyticus, and the deletion strain increased the growth rate of Vibrio alginolyticus. RseB gene deletion resulted in a 37.5% decrease in hemolysis of V. alginolyticus. The pathogenicity test results showed that the death time of the RseB gene deleted strain in the experimental group was delayed by 4 h, and the LT₅₀ was delayed by 20 h. At 24 h after challenge, the colonization ability of RseB gene deletion strain in L. vannamei decreased by 53.4%. The hepatopancreas of L. vannamei infected by Vibrio phagolysii were seriously infected, the order of hepatic tubules was out of order, the space became larger, the boundary began to blur gradually, and the lumen of hepatopancreas tubules disappeared. The arrangement of liver cells began to be irregular, and some liver cells were obviously enlarged or even the whole cell began to degenerate, dyed dark with eosin, and the nucleus began to disappear. The results of pathological sections at 8, 16, 32 and 64 h showed that the hepatopancreas damage of RseB gene knockout strain was relatively mild.