Abstract: In this study, scFv-coding genes were obtained from a hybridoma cell and expressed in Bac-to-Bac baculovirus expression system, and the characterizations of scFv were verified by Western blotting, ELISA and compared with monoclonal antibody. Firstly, the specific heavy and light variable chains were amplified from hybridoma cell line 4C6, which secreting monoclonal antibody(mAb) against parathion-ethyl, and then they were assembled as scFv fragments by splice overlap extension polymerase chain reaction(SOE-PCR). Thereafter, the bacmid-scFv-4C6 was constructed and transfected into Sf9 insect cells to generate the recombinant antibody which was further verified by Western blotting. The results demonstrated that the recombinant scFv-4C6, with the relative molecular mass of approximately 28.3 kD, was successfully constructed and expressed in Sf9 cells. Besides, the expression peak was reached at 72 h after the transfection. As tested by ic-ELISA, the performances of scFv-4C6 were similar to those of the parental mAb, with a high sensitivity(IC50 value) of 7.9 ng/mL to parathion-ethyl, 12% cross reactivity to parathion-methyl, and 1.8% cross reactivity to fenitrothion.