This study was carried out to determine the level of Chlorpyrifos (CPF), an organophosphorus insecticide, in water of Abbassa and Sahl El-Hussinia fish farms and investigate the sub lethal concentration of this pesticide on immune response of Oreochromis niloticus (O. niloticus). Water samples were collected from Abbassa and Sahl El-Hussinia fish farms and analyzed using Gas chromatograph (GC) for detection of Chlorpyrifos level. Two hundred and ninety O. niloticus with average body weight (35±0.5 g) were collected from Abbassa fish hatchery, Sharkia Governorate, Egypt. One hundred and seventy of these fish were used for the determination of acute toxicity of Chlorpyrifos, while, another 120 fish were used for the determination of the effect of different sub lethal concentrations of Chlorpyrifos (1/8, 1/20 and 1/43 of 96 h LC50) on the immune response of O. niloticus. It was found that; the levels of Chlorpyrifos in Abbassa and Sahl El-Hussinia fish farms were 0.008 and 0.0016 mg/L, respectively. The LC50 value of Chlorpyrifos was 0.07mg/L. Sub-lethal concentration of Chlorpyrifos altered the non-specific immunological parameters (namely, total globulin, immunoglobulin M (IgM), lysozyme, nitric oxide, phagocytic activity) and interlukin-1β (IL-1β) of O. niloticus. It could be concluded that water of Abbassa and Sahl El-Hussinia fish farms have had detectable residue levels of CPF, which was altered the immunological status of O. niloticus.