Abstract: The use of probiotics is one of the non-antibiotic methods of preventing and controlling bacterial infections in aquaculture. Lactobacillus acidophilus can secrete antimicrobial substances in the body, which can inhibit the growth of harmful bacteria, regulate the micro-ecological balance of intestinal tract, and enhance the resistance to toxicity and disease. We analyzed the growth of L. acidophilus, Vibrio parahaemolyticus, V. harveyi and V. coralliilyticus under different oxygen environments and different media conditions, and systematically evaluated the antagonistic effect of L. acidophilus on the above mentioned Vibrio species, as well as the composition and changes of the intestinal flora of Litopenaeus vannamei, by using Oxford cup method and liquid culture method in addition with PCR fluorescence quantification and high throughput sequencing technology. The results show that anaero bic environment promoted the growth of V. harveyi significantly (P<0.05). Different ratios of media had a significant effect on the growth of all the four bacteria. L. acidophilus inhibited the growth of all the three Vibrio species effectively (P<0.05). In the background of intestinal flora of prawns, L. acidophilus inhibited the growth of V. parahaemolyticus significantly (P<0.05). The proportion of Vibrio spp. decreased significantly in the bacterial solution and supernatant groups compared with the control group. Beneficial genera of bacteria increased significantly in the high bacterial concentration group. The results indicate that L. acidophilus inhibits the growth of the above three Vibrio species in plate and liquid cultures, and inhibits the growth of V. parahaemolyticus in the background of prawn intestinal microbiota, being able to optimize the structure of microbiota.