An 8-week feeding experiment was conducted to quantify the dietary isoleucine requirement of fingerling Indian major carp, Labeo rohita (3.50 ± 0.04 cm; 0.40 ± 0.02 g) using amino acid test diets (400 g kg−1 crude protein; 17.90 kJ g−1 gross energy) containing casein, gelatin and l-crystalline amino acids. Six dietary treatments supplemented with graded levels of isoleucine (7.5, 10.0, 12.5, 15.0, 17.5 and 20.0 g kg−1), in gradations of 2.5 g kg−1 diet, were fed to triplicate groups of fingerlings to apparent satiation divided over two feedings at 07:00 and 17:30 h. Performance of the fish was evaluated on the basis of live weight gain, feed conversion ratio (FCR), protein efficiency ratio (PER), specific growth rate (SGR) and protein productive value (PPV). Statistical analysis of live weight gain, FCR, PER, SGR and PPV reflected significant differences among treatments. Live weight gain and conversion efficiencies were best with isoleucine at 15.0 g kg−1 of diet. Live weight gain, FCR, PER, SGR and PPV data were also analysed using second-degree polynomial regression analysis to obtain more accurate isoleucine requirement estimate which was found to be at 15.9, 15.3, 15.2, 15.8 and 15.7 g kg−1 of dry diet, corresponding to 39.8, 38.3, 38.0, 39.5 and 39.3 g kg−1 of dietary protein respectively. Based on the quadratic regression analysis of the live weight gain, FCR, PER, SGR and PPV, the optimum level of isoleucine for fingerling L. rohita is in the range of 15.2–15.9 g kg−1 of dry diet, corresponding to 38.0–39.8 g kg−1 of dietary protein. Maximum body protein, minimum moisture and fat were noted at 15.0 g kg−1 of dietary isoleucine while the body ash remained constant among all the treatment levels. No mortality was recorded during the duration of the experiment.