This experiment was designed to investigate the effects of dietary valine on the growth performance, feed utilization, digestive enzymes, serum antioxidant and immune indices of juvenile Trachinotus ovatus and determine its valine requirement. Six diets with different concentrations of L‐valine (15.0, 16.6, 18.6, 20.7, 23.5 and 25.4 g/kg dry diet, defined as diet Val‐1 to Val‐6.), were formulated to contain 430 g/kg crude protein with fish meal, soybean meal, peanut meal and precoated crystalline amino acids. Each diet was randomly assigned to triplicate treatments of 20 fish (the initial body weight was 5.34 ± 0.03 g) for 8 weeks. The results indicated that the final body weight and percent weight gain (PWG) increased with increasing valine concentration up to 18.6 g/kg (diet Val‐3), whereas the diets containing higher valine concentration reduced the growth performance significantly (p < .05). Moreover, the protein efficiency ratio, body protein deposition (BPD), muscle protein content, intestinal amylase and pepsin activities, serum T‐AOC, LZM activities, IgM, complement 3 and complement 4 concentration had a similar trend with PWG, and the trend of feed conversion ratio, serum AST, ALT activities, urea and MDA content was opposite. Meanwhile, the lipid contents of whole fish and muscle in diet Val‐6 were particularly lower than other diets (p < .05). The survival rate of diet Val‐1 was lowest in this study and was significantly lower than diet Val‐2 (p < .05). The results of polynomial regression based on PWG and BPD indicated that the optimal dietary valine requirement for Trachinotus ovatus reared in seawater‐floating net cages was 19.87–20.17 g/kg valine of dry diet, correspondingly 46.22–46.91 g/kg of dietary protein.