A 2×2 factorial design was conducted to elucidate the effect of dietary biotin on growth performance, body composition, tissue lipid content and lipid metabolism related genes expression of blunt snout bream, Megalobrama amblycephala fed with different dietary lipid levels. Juvenile fish (average initial weight: 36.0±1.0g) were fed with diets containing two lipid levels (5% and 11%) and two biotin levels (0.06mgkg−1 and 2.50mgkg−1) for 8weeks, respectively. Specific growth rate (SGR), hepatosomatic index (HSI), mesenteric fat index (MFI), hepatopancreas lipid, total triacylglycerol (TG) and cholesterol (TC) contents, plasma TG and muscle TG contents were significantly (P<0.05) affected by dietary lipid. No significant interactions between lipid and biotin were observed. Significant (P<0.05) interactions between lipid and biotin were observed in muscle lipid, hepatopancreas saturated fatty acids (SFA) and muscle EPA contents. Hepatopancreas monounsaturated fatty acids (MUFA), polyunsaturated fatty acids (PUFA) and EPA+DHA contents were significantly (P<0.05) affected by dietary biotin. At dietary lipid levels of 5 or 10%, hepatopancreas MUFA contents were decreased but PUFA and EPA+DHA contents were increased significantly (P<0.05) with dietary biotin levels increase. Significant (P<0.05) interaction between lipid and biotin were also observed in hepatopancreas acetyl-CoA carboxylase α (ACCα), sterol regulatory element-binding protein 1 (SREBP1), acyl-CoA oxidase (ACO) and acyl-CoA oxidase (FACS) genes expression levels. ACCα, fatty acid synthetase (FAS), liver X receptor α (LXRα), SREBP1, ACO and FACS genes expression levels significantly (P<0.05) increased with the increasing dietary biotin levels. ACCα, peroxisome proliferatoractivated receptor α (PPARα), carnitine palmitoyltransferase I(CPTI) and ACO genes expression levels significantly (P<0.05) decreased with the increasing dietary lipid levels. In conclusion, no effects of biotin on growth performance, body parameter, whole-body composition in fish fed with 5% or 11% lipid diets were found. Dietary biotin could play a role in hepatopancreas fatty acid synthesis and β-oxidation related genes expression. Dietary biotin could promote hepatopancreas polyunsaturated fatty acids synthesis of fish.