The present research was designed to study and compare the biochemical parameters in Labeo rohita due to exceptional nutritional value of this fish as a protein source in developing countries. The protein and heavy metal (Zn, Cu, Ni) activities were determined spectrophotometrically. The proximate analysis of L. rohita collected from different localities of Pakistan revealed that the protein (19.97%) and ash contents (1.76%) were highest in hatchery L. rohita while the fat (0.84%), carbohydrate (5.39%) and dry matter contents (24.11%), were maximum in the river L. rohita. In comparison to the hatchery and river L. rohita, the moisture contents (81.42%) were the highest in farmed fish. During enzymatic analysis, maximum activities of the peroxidase, α- amylase and mutarotase were recorded in hatchery fish whereas the catalase and superoxide dismutase activities were found to be the highest in farmed fish. The maximum accumulation of Cu and Ni metals were observed in hatchery fish and least in farmed fish and the highest Zn accumulation was observed in river fish. Analysis of variance on catalase, peroxidase, α-amylase activity and metal accumulation showed statistically significant differences at p<0.05 among sampling sites and fish organs and interaction between sampling sites and fish organs.