The Chinese sturgeon (Acipenser sinensis), a flagship species of the Yangtze River and national first-class key wild protected animal, currently relies on stock enhancement and release as the most effective and direct means of population conservation. However, juveniles from full artificial propagation exhibit weaker physical conditions than those from wild populations, including slow growth, low feeding efficiency, and poor stress resistance. To investigate the effects of dimethyl-β-propiothetin (DMPT) supplementation on growth, serum immunity, intestinal digestive enzyme activities, and gut microbiota composition of Chinese sturgeon juveniles, a 5-week feeding trial was conducted with sub-adult second-generation juveniles (initial body weight: 300.88 ± 17.64 g). DMPT was added to the basal diet at 0, 400, 800, and 1600 mg/kg (CK, A1, A2, and A3 groups, respectively), with 3 replicates per group and 10 fish per replicate. The results showed that weight gain and specific growth rate (SGR) in groups A1, A2, and A3 were significantly higher than those in the CK group. The feed conversion ratio in group A3 was significantly lower than that in the CK group. The contents of serum immunoglobulin M (IgM), complement C3, complement C4, and lysozyme activity increased progressively with increasing DMPT levels, with values significantly higher in groups A2 and A3 than those in the CK group. Serum superoxide dismutase activity was significantly lower in all treatment groups than that in the CK group, while alkaline phosphatase activity was significantly higher in groups A2 and A3. Digestive enzyme activities exhibited a trend of initially increasing and subsequently decreasing with increasing DMPT levels. Protease and lipase activities in groups A1 and A2 were significantly higher than those in the CK group, while amylase activity in all treatment groups was significantly higher than that in the CK group. No significant differences were observed in ACE, Chao1, Shannon, or Simpson indices among the treatment and control groups; however, the phylogenetic diversity whole tree (PD_whole_tree) index in groups A2 and A3 was significantly higher than that in the CK group. At the phylum level, Fusobacteria was the absolute dominant gut phylum across all groups, followed by Proteobacteria and Firmicutes; Fusobacteria relative abundance initially decreased and subsequently increased with increasing DMPT levels. At the genus level, Cetobacterium was the absolute dominant genus, with relative abundance progressively increasing with higher DMPT supplementation. The secondary dominant genera in groups CK and A2 were Plesiomonas and Escherichia-Shigella, while those in A1 and A3 groups were Plesiomonas and Bacteroides. Liver gene expression of growth hormone receptor (GHR) in groups A2 and A3 and insulin-like growth factor-1 (IGF-1) and IGF-2 in group A3 were significantly higher than that in the CK group, with GHR, IGF-1, and IGF-2 expression increasing progressively with DMPT dosage. In conclusion, appropriate DMPT supplementation significantly improved growth performance, immune capacity, and digestive capacity in juvenile Chinese sturgeon. Among the four addition gradients (0, 400, 800, and 1600 mg/kg) established in this study, 1600 mg/kg was optimal for promoting growth and health. This dosage can effectively enhance liver growth-related gene expression, thereby promoting the growth of the fish; simultaneously, its key immune indicators were significantly superior to those of the control and low-dose groups. Although DMPT supplementation did not affect species richness or diversity of intestinal flora, it increased the relative abundance of dominant phyla and genera, thereby contributing to improved growth, intestinal health, and disease resistance.