In this experiment, two strains of probiotics, i.e. Enterobacter hormaechei (E3) and Lactobacillus (R3) were used to feed Litopenaeus vannamei for 4 weeks. The diversity of intestinal microflora of L. vannamei in experimental group and blank group was compared and analyzed by Biolog-ECO method in the later period of feeding, in order to evaluate the effect of probiotics on the metabolic function of intestinal microflora of L. vannamei. The results showed that AWCD in the experimental group was significantly higher than that in the blank group, indicating that probiotics enhanced the intestinal microbial activity; the ability of intestinal microorganisms to utilize carbon source was significantly enhanced, which indicated that the digestive enzyme secreted by probiotics increased the digestibility and absorption rate of prawn feed, thus promoting the rapid growth of L. vannamei. There were significant differences in intestinal microbial diversity index (including Shannon, Simpson and McIntosh indexes), which indicated that different strains had different effects on intestinal microflora richness of L. vannamei. The results of sampling on the 1st and 5th day after stopping adding E. hormaechei showed that Shannon index decreased significantly and Simpson index and McIntosh index increased significantly, and lactic acid bacteria was stopped from adding. The results of sampling on the 1st and 5th day showed that there was no significant difference in Shannon index and McIntosh index, but there was no significant difference between them on the 10th day. The results showed that the addition of probiotics to the feed could change the number and structure of the original microflora in the intestinal tract of L. vannamei and promote the complex interaction between the microbial communities in the intestinal tract of L. vannamei. In turn, it plays an important role in maintaining or promoting the health of L. vannamei. It also showed that the two probiotics should stay in the intestines of aquatic animals for at least 5 days.