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According to the common structure of three organophosphorus pesticides (Isocarbophos, Isofenphos-methyl and Isofenphos), hapten (H1) was synthesized.Bovine serum albumin (BSA) and ovalbumin (OVA) were used as protein carriers respectively to couple with H1 by an active ester method.Complete antigens H1-BSA and H1-OVA were prepared as immunogen and coating antigen, respectively.At last, one cell strain of monoclonal antibody was obtained and named 6B5/E3, which can beused to detect the three pesticides.After optimization, the standard curves of these three pesticides were established and the detection limits of the three pesticides were 12.19, 34.51 and 19.68 ng/ml, respectively.In addition, other six kinds of organophosphorus pesticides were tested by using this assay and the results were regarded as negligible.The recoveries obtained by the three standard pesticides addition to real samples, including rice, water and soil, were all from 91.63% to 112.86%.Therefore, the optimized ELISA might become a convenient and satisfied analytical tool for monitoring Isocarbophos, Isofenphos-methyl and Isofenphos residues in real samples.