To study the effect of sanguinarine on intestinal microbiota diversity in Trionyx sinensis, 480 individuals [initial weight (31.75±7.20) g] were randomly selected and divided into four groups. Three experimental diets RS2, RS3, and RS4 were formulated with 50, 100, and 150 mg/kg sanguinarine, respectively, and no sanguinarine was included in the control group. At the end of the experiment, the total DNA of the midgut tissue was extracted and the 16S rRNA V3-4 region was sequenced. The results showed that the total number of operational taxonomic units (OTU) in experimental groups was significantly higher than that in the control group, and the complexity and richness of intestinal flora was also significantly higher than that of the control group. Analysis of the structure and composition of the intestinal flora revealed that RS1 and RS2 were closer in structure, whereas RS1 was farther away from RS3 and RS4. Proteobacteria and Chlamydiae were the dominant phyla, and Helicobacter was the dominant genus. Helicobacter abundance in experimental groups was significantly lower than that in the control group, whereas the abundance of Acinetobacter, Lactobacillus, and Bifidobacterium were all significantly higher than that in the control. Therefore, the study demonstrated that adding an appropriate amount of sanguinarine (100~150 mg/kg) in the feed could effectively promote intestinal flora diversity and richness, and improve the community structure of T. sinensis intestinal flora.