Abstract: The hapten for the insecticides chlorpyrifos was prepared starting from PSCl3 via three steps. Then,the hapten was conjugated to bovine serum albumin (BSA) with CDI method and ovalbumin (OVA) with mixed anhydride method to form the immuno-antigens and the coating antigen,respectively. The conjugation ratio was 14.6 and 6.2 for the hapten-BSA conjugate and the hapten-OVA conjugate. The polyclonal antibody (PAb) was obtained with high titer (2.56×104 for antiserum and 2.56×106 for purified antibody) after immuning to rabbits. The specificity studies showed that the cross-reactivities were all less then 4%,which indicated that the immunoassay was quite specific for chlorpyrifos.The affinity studies showed that the real detection limit of the ELISA,for standards,was 1.0 ng/mL with an I50 value of 8.2 ng/mL. The linear regression equation for the assay was described as follow: y=23.503 lg(x)+28.556 (r=0.991 9),with the linear ranging form 1 to 500 mg/mL.