Abstract: The leopard coral grouper (Plectropomus leopardus) is a marine aquaculture species of high economic value. However, issues such as nutritional fatty liver disease, frequent disease outbreaks, and enteritis have severely hindered the healthy development of the P. leopardus aquaculture industry. Bile acids are a class of amphipathic compounds synthesized in the liver from cholesterol, they are not only involved in the digestion and absorption of lipids but also play crucial roles in regulating lipid metabolism, maintaining intestinal homeostasis, and enhancing immune defense. Previous studies have demonstrated the beneficial physiological effects of bile acids in various aquatic animals. Nevertheless, the composition and characteristics of bile acids in the liver and serum of the P. leopardus have not been reported. To elucidate the composition and tissue distribution of bile acids in P. leopardus, ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was employed to detect and analyze 50 bile acid species in the liver and serum. The results revealed that 26 bile acids were detected in the liver of P. leopardus, including 6 primary bile acids: taurocholic acid (TCA), chenodeoxycholic acid (CDCA), taurochenodeoxycholic acid (TCDCA), glycochenodeoxycholic acid (GCDCA), glycocholic acid (GCA), and cholic acid (CA). Taurine-conjugated bile acids were the predominant bile acid species in the liver, with TCA and TCDCA being the most abundant, together accounting for 91.99% of the total hepatic bile acid pool. The remaining 20 were secondary bile acids, among the detected secondary bile acids, ω-muricholic acid & α-muricholic acid & 3β-cholic acid (ω-MCA & α-MCA & 3β-CA) exhibited the highest concentration, while isoallolithocholic acid (isoALCA) showed the lowest. In the serum, 21 bile acids were identified, including the same 6 primary bile acids. TCA, TCDCA were the most abundant primary bile acids, whereas GCA was the least abundant. Among the secondary bile acids in serum, glycodehydrocholic acid (GDHCA), 7,12-diketolithocholic acid (7,12-DKLCA), and ω-MCA & α-MCA & 3β-CA were present at the highest levels, while 7-ketodeoxycholic acid (7-KDCA) was the lowest. This study demonstrates that the bile acid profiles in the liver and serum of P. leopardus are dominated by primary bile acids such as TCA, CDCA, and TCDCA, but also contain various secondary bile acids including ω-MCA & α-MCA & 3β-CA, GDHCA, and 7,12-DKLCA. The composition of bile acids in the liver and serum of P. leopardus was systematically characterized, clarifying the types and concentrations of bile acids in these two tissues. These findings enrich the bile acid atlas of fish and provide precise data to support the future application of bile acids in this species.