Abstract: Carbohydrate is an important non-protein energy source in aquaculture. Adding an appropriate amount of carbohydrate to feed not only spares dietary protein and improves its utilization, but also reduces ammonia-nitrogen excretion and water pollution. However, excessive carbohydrate inhibits growth, induces hepatobiliary disorders and lowers stress tolerance, ultimately impairing production efficiency. Previous studies indicated that dietary bile acid supplementation can modulate glycolipid metabolism and alleviate high-carbohydrate-induced disturbances, yet the long-term effects of high-carbohydrate feeding on the bile acid profile of fish have not yet been examined. This study determined the bile-acid spectrum of Megalobrama amblycephala and evaluated its response to long-term high-carbohydrate feeding, thereby clarifying mechanisms underlying carbohydrate-associated hepatobiliary syndromes and informing nutritional interventions. A total of two experimental diets were prepared, including a control diet (29% nitrogen-free extract) and a high-carbohydrate diet (HC, 41% nitrogen-free extract). Each diet was fed to four replicates of juvenile M. amblycephala (35.20 ± 0.15) g for 12 weeks. Then, the bile acid spectrum of M. amblycephala was analyzed in the liver, gallbladder, hindgut and plasma using the high-throughput target quantitative detection technology. The results showed that both taurochenodeoxycholic acid (TCDCA) and taurocholic acid (TCA) are the main bile acids in M. amblycephala, and there was a significant positive correlation between them. Compared with the control group, the contents of taurolithocholic acid-3-sulfate (TLCA-3S) increased significantly in the liver of the HC group. The contents of chenodeoxycholic acid (CDCA), taurolithocholic acid-3-sulfate (LCA-3S), glycocholic acid (GCA), TCDCA, TCA, chenodeoxycholic acid (ACA) and taurodeoxycholic acid (TDCA) all decreased significantly in the liver of the HC group. The contents of TCDCA and TCA both increased significantly in the gallbladder of the HC group, while that of LCA-3S and lithocholic acid (LCA) both decreased significantly. The levels of LCA increased significantly in the hindgut of the HC group, while that of cholic acid (CA), LCA-3S, GCA, TCDCA, TCA and glycodeoxycholic acid (GDCA) all decreased significantly. Furthermore, the contents of cholic acid (CA), LCA and TLCA-3S all increased significantly, while that of chenodeoxycholic acid 24-acyl-β-D-glucuronide (CDCA-24Gln) decreased significantly. In addiiton, glycine-conjugated bile acids were found in the tissues of M. amblycephala, including GCA, glycochenodeoxycholic acid (GCDCA) and glycoursodeoxycholic acid (GUDCA). Furthermore, tauro-α-muricholic acid (T-α-MCA) was first found in fish. In conclusion, both TCDCA and TCA are the main bile acids in M. amblycephala, and there was a significant positive correlation between them. A long-term feeding of the HC diet significantly decreased the total bile acid content, and altered the component of bile acids.