In this study, we investigated the effects of Clostridium butyricum (group A), Bacillus subtilis (group B), and the immune enhancer algal β-1,3 glucan (group C) on the intestinal flora of Mauremys reevesii and the effects of C. butyricum on the transcriptome of M. reevesii splenic immune tissues. M. reevesii were assigened to four groups, each containing three replicates from 18 samples. Juvenile turtles with an initial weight of 106.35 ± 0.03 g were fed a basic diet containing no probiotics (control group D), or a basic diet containing the A, B, or C supplement. After the turtles had been fed for 60, 90, and 120 d of the experimental period, high-throughput sequencing of the 16S rRNA gene revealed no significant difference in alpha diversity among the four groups at 60 days of feeding (P > 0.05), and at 90 days, the alpha diversity in group A was significantly different (P < 0.05), with an increase of 26.62% in the Shannon index and a decrease of 83.33% in the Simpson index; at 120 d, the alpha diversity (Shannon index)showed a decreasing trend in order for groups A, B, and C, At the phylum level, the abundance of Bacteroidetes, Proteobacteria, and Fusobacteria in group A increased significantly with increasing feeding time (P < 0.05),At the genus level, the abundance of Ruminococcaceae and Anaerotruncus in group A increased significantly compared with that in the other three groups (P < 0.05). Transcriptome analysis showed that 384 genes were differentially expressed in the spleen of M. reevesii, 195 genes were upregulated and 189 genes were downregulated, and C. butyricum TF201120 regulated the hematopoietic cell lineage signaling pathway in the spleen of M. reevesii (P < 0.05). The regulation of several identified immune-related genes was confirmed by qPCR, These results showed that C. butyricum, B. subtilis and the immune enhancer algal β-1,3 glucan can improve the intestinal flora of M. reevesii, with C. butyricum TF20 being the most effective and significantly enhancing the immunity of M. reevesii.