An engineering anti-human TNF-α single-chain antibody (ScFv) gene was cloned into the expression vector pET15b-Etag, and expressed in E.coli BL21(DE3) under the control of T7 promoter. By using 0.1 mmol/L IPTG induction, the amount of the ScFv expression product was more than 38% of total bacterial proteins. Most of them existed in a form of inclusion body. More than 6% of total bacterial proteins were soluable and can be detected in the part of periplasm, which can bind with rhuTNF-α by ELISA and Dot-blotting technique.