Jatropha protein isolate was prepared following the principle of isoelectric precipitation. The isolated protein is low in fibre, lipid and ash, and high in protein and gross energy contents. The antinutritional factors such as phytic acid, tannin and trypsin inhibitor were found to decrease after protein extraction except phorbol esters (PEs), which remain bounded to the protein matrix. Thus, the protein isolate was detoxified and the PEs content reduced significantly. For the feeding trial, six isonitrogenous diets were formulated as DJP 0, DJP 25, DJP 50, DJP 75, DJP 100, or DJP 100 + lysine with detoxified Jatropha protein isolate (DJP) in replacement for soybean protein isolate (SPI) and fed to L. rohita fingerlings (3.25 ± 0.02 g) for 60 days. The weight gain percentage (WG %), specific growth rate and metabolic growth rate values among the groups showed no significant difference (p > .05), except DJP 100 fed group. The feed conversion ratio, protein efficiency ratio, protein productive value, and survival, digestive and hepatic aminotransferase enzymes activities were not significantly affected, whereas intestinal alkaline phosphatase and serum alanine transaminase differ significantly (p < .05). Thus, this study revealed that DJP supplemented with lysine could replace 100% SPI protein without causing any significant effects on fish performance and organ integrity.