To construct the recombinant vector pBV220-scFv and express anti-clenbuterol (CBL) scFv antibody in Escherichia coli, we amplified the scFv gene using plasmid pCANTABSE-CBL as a template, recombined it with pPICZalphaA, then amplified the scFv-His-tag gene from plasmid pPICZalphaA-scFv and linked it with expression plasmid pBV220. We identified the recombinant plasmid by restrictive enzyme digestion, PCR amplification and sequence analysis. Finally, we transformed the recombinant vector into E. coli DH5alpha that was temperature-induced and expressed recombinant protein. We identified the recombinant protein by SDS-PAGE, Western blotting and indirect competitive ELISA. The results show that recombinant plasmid pBV220-scFv contained the inserted fragment with highest homology about 99.8%. The expression of scFv induced by temperature show 37 kD Mw and anti-His-tag mAb recognized-activity by SDS-PAGE and Western blotting respectively, and could competitively combine with CBL, the IC50 is 4.55 ng/mL. The recombinant plasmid pBV220-scFv is constructed and expresses the scFv gene of CBL in E. coli successfully. This study suggests the corresponding immunoassay methods could be set up by the recombinant scFv.