The present study was under taken to assess the toxicological effect of Malathion (an Organophosphate) on electrophoretetic banding patterns of esterase enzymes in gill and intestine tissues of freshwater fish Labeo rohita (Hamilton) at different time intervals i.e. 24H, 48H, 72H and 96H and was compared with control. The esterase isozymes were quantitatively analyzed by using 7.5٪ native polyacrylamide gel electrophoresis (PAGE) stained with α-naphthyl acetate as substrate. Three different esterase enzyme bands were detected and named as Est-1; Est-2 and Est-3 with different relative mobilities such as 0.6± 0.05; 0.4± 0.05; 0.3± 0.05 in gill and intestine tissue. All the three esterase bands were found in gill and intestine tissues in control. After the fish is exposed to Malathion (an Organophosphate), in gill tissue Esterase-1 shown resistance, Esterase-2 and Esterase-3 were severely affected, where as Esterase-3 was severely affected than Esterase-2 and Esterase-1 exhibited resistance in Intestine tissue.