The objective of this study was to develop an enzyme-linked immunosorbent assay (ELISA) with specific monoclonal antibodies for quick detection and screening of a broad spectrum of O,O-diethyl organophosphorus pesticides. Diethylphosphono acetic acid was used as a hapten and conjugated with bovine sera albumin (BSA) or ovalbumin (OVA) to prepare immunogen and coating antigen, respectively. Two hybridoma cell lines that produce monoclonal antibodies against organophosphorus pesticides have been obtained by fusing mouse myeloma cells SP2/0 and splenocytes from Balb/c mice immunized with the immunogen.The immunoglobulin class was analyzed by double immunodiffusion and the affinity and specificity of McAbs were determinated with indirect enzyme-linked immunosorbent assay (ELISA). Results indicated that the immunoglobulin class of McAb was IgG1, McAbs have a high affinity for diethylphosphono acetic acid (1.4×10⁷ L/mol) and are able to cross-react to Chlorpyifos, Parathion, Profenofos, Omethoate, Dichlofenthion, Diazinon, Bromophos, Phoxim. This immunoassay is therefore capable of detecting 8 different organophosphorus pesticides. It is concluded that this assay is a qualified screening tool for quantitative or semi-quantitative detection of the 8 organophosphorous pesticides.