This study was conducted to determine the optimal dietary biotin requirement of juvenile Megalobrama amblycephala. Quadruple groups of fish (initial average weight 2.01 ± 0.01 g) were fed thrice daily with six isonitrogenous and isoenergetic purified diets containing 0 (basal diet), 0.015, 0.049, 0.158, 0.624 and 2.49 mg kg−1 biotin, respectively, for 8 weeks. Results showed that survival rate, final weight, feed intake, specific growth rate, protein efficiency ratio and nitrogen retention efficiency all increased significantly (P < 0.01) as dietary biotin levels increased from 0 to 0.049 mg kg−1, whereas the opposite was true for feed conversion ratio. Dressout percentage, condition factor, hepatosomatic index, viscera/body ratio all showed no significant difference (P > 0.05) within the biotin range tested. Contrary to moisture content, whole-body protein and lipid contents showed a positive correlation with dietary biotin levels. In addition, liver biotin content increased significantly (P < 0.05) with increasing dietary biotin levels up to 0.624 mg kg−1. Hepatic pyruvate carboxylase (PC) and acetyl-CoA carboxylase (ACC) activities both showed an increasing trend as dietary biotin levels increased. Based on the regression analysis of weight gain, hepatic PC and ACC activities, the optimal dietary biotin requirement of juvenile Megalobrama amblycephala is estimated to be 0.063, 0.071 and 0.075 mg kg−1, respectively.