Abstract: Objective: To expect more efficient expression, we expressed scFv CL-3, which specifically targets to human colon cancer membrane antigen, as inclusion bodies in E.Coli. Methods: Recombinant plasmid pJW2 CL-3 were transformed into E.Coli DH5 α. Induced by temperature control, scFv CL-3 were expressed as inclusion bodies in the plasms of bacteria when the positive clone has been cultured in LB till OD600=0.6. After being digested and sonicated, inclusion bodies were dissolved in 8M urea and then diluted 100 fold into renaturation buffer at 10℃. The scFv were purified on ion exchange Blot. Immunoactivity of the scFv was appraised by ELISA. Results: After 5 hour inducing, the inclusion bodies turn out to account for about 30% of total bacterial proteins. Inclusion bodies were dissolved in 8M urea and have been refolded in renaturation buffer after 48 hours at 10℃. When eluted with 0.3 mol/L NaCl/20 nmol/L TE, scFv were obtained from ion exchange column elution. SDS PAGE and Western Blot proved that the 27KD protein expressed in E.Coli is scFv with E-tag fused to its Cterminus. The renatured scFv CL-3 has similar specificity and Immunoactivity to its parent antibody as evaluated by ELISA. Conclusion: Expressed scFv CL-3 as inclusion bodies in E.Coli. After renaturation and purification, the scFv were showed could bind specifically to human colon cancer antigen.