Abstract: In this study, chemiluminescence enzyme-linked immunoassay (CLEIA) for the detection of the three organophosphorus pesticides was established and optimized. A monoclonal antibody with broad-specificity to parathion, parathion-methyl, and fenitrothion was employed. The direct and indirect competitive CLEIAs (dc-CLEIA and ic-CLEIA) were compared, and the physical and chemical parameters in those two reaction models were optimized. The half-maximum inhibition concentration (IC₅₀) and the linear range from ic-CLEIA was respectively 5.57 μg/kg and 0.39-100 μg/kg for parathion, 2.30 μg/kg and 0.39-25 μg/kg for parathion-methyl, and 2.62 μg/kg and 0.39 to 25 μg/kg for fenitrothion. The half-maximum inhibition concentration (IC₅₀) and the linear range for dc-CLEIA was respectively 5.43 μg/kg and 0.39-100 μg/kg for parathion, 1.34 μg/kg and 0.10-25 μg/kg for parathion-methyl, and 1.24 μg/kg and 0.10-25 μg/kg for fenitrothion. The novel methods for the detection of the above mentioned organophosphorus pesticides can meet the requirements of their maximum residue limits in fruits and vegetables, which paves the way to produce rapid-test kits for the detection of organophosphorus pesticides.