Tributyrin (TB), as a novel feed additive, holds broad market prospects and is crucial for promoting fish growth and maintaining intestinal health. We first identified the fatty acid metabolism-related gene cpt1b in the intestines of mandarin fish (Siniperca chuatsi) from the TB-supplemented group. A total of 600 mandarin fish (200.0 ± 5.0 g) were evenly allocated into three groups. The control group (C) received only the standard extruded feed, while the experimental groups were supplemented with tributyrin (TB) at concentrations of 500 mg/kg (T1 group) and 1000 mg/kg (T2 group), respectively. Cloning yielded a 2364 bp open reading frame (ORF) encoding 787 amino acids, with the gene possessing two conserved transmembrane domains. Phylogenetic analysis further indicated a close phylogenetic relationship between largemouth blackbass (Micropterus salmoides) and mandarin fish. Tissue distribution and intestinal enzyme activity analyses revealed that supplementation with varying concentrations of TB upregulates cpt1b gene expression in different tissues, while modulating intestinal digestive enzyme and antioxidant enzyme activities. Our findings suggest a potential mechanism involving enhanced intestinal enzyme activity, reduced fat accumulation, increased expression of lipid oxidation-related genes, and accelerated TB degradation in the intestine.