An 8-week feeding experiment was conducted to evaluate the dietary leucine requirement of fingerling Indian major carp, Labeo rohita (3.50±0.04 cm; 0.40±0.02 g) using amino acid test diets (40% crude protein; 17.90 kJ g−1 gross energy) containing casein and gelatin as intact protein sources and l-crystalline amino acids. Growth performance and biochemical parameters were assessed by feeding six amino acid test diets supplemented with graded concentrations of leucine (0.75, 1.0, 1.25, 1.50, 1.75 and 2.0 g per 100 g) to triplicate groups of fingerlings to apparent satiation divided over two feedings at 07:00 and 17:30 hours. Performance of the fish was evaluated on the basis of live weight gain, feed conversion ratio (FCR), protein efficiency ratio (PER) and body protein deposition (BPD) data. Maximum live weight gain (315%), best FCR (1.35), highest PER (1.86) and BPD (33.9) were recorded at 1.50 g per 100 g dietary leucine. Statistical analysis of live weight gain, FCR, PER and BPD data reflected significant differences (P<0.05) among treatments. Live weight gain, FCR, PER and BPD data were also analysed using second-degree polynomial regression analysis to obtain more accurate leucine requirement estimate which was found to be at 1.57, 1.55, 1.52 and 1.50 g per 100 g of dry diet, corresponding to 3.92, 3.87, 3.80 and 3.75 g per 100 g of dietary protein respectively. Based on the quadratic regression analysis of the live weight gain, FCR, PER and BPD data, the optimum requirement of fingerling L. rohita for leucine is estimated to be in the range of 1.50–1.57 g per 100 g of the dry diet, corresponding to 3.75–3.92 g per 100 g of dietary protein.