The effects of isolation on the growth performance, behavior, blood hematology, and cortisol level of Nile tilapia, Oreochromis niloticus were checked in laboratory conditions. All fishes were closely observed for behavior and growth-related parameters. The weight and diameter of both the control and treatment groups were measured before and after the trial of 28 days using an electronic balance and measuring tape, respectively. At the end of the trial, blood was drawn from the caudal fin to determine some hematological and hormonal parameters. Significant differences were seen in growth performance, cortisol level, and hematology, as weight gain, specific growth rate, relative growth rate, and daily food intake were significantly higher in the control group. In comparison, cortisol levels and feed conversion ratio were significantly higher in the isolated group. White blood cells, lymphocytes, red blood cells, hemoglobin, hematocrit, and mean platelet volume, were significantly higher in the isolated group when compared to the control group. At the same time, the amount of granulocytes, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, and platelets was significantly higher in the control group. Isolated fish showed aggressive behavior towards introduced fish from the control group. Isolation increased the cortisol level, which indicates that the fish was stressed. It is concluded that isolation significantly affects the growth performance, cortisol level, and hematology of O. niloticus.