Objective: To make a fusion protein constructed of human anti-EGFR scFv/truncated protamine(scFv/tP) gene and analyze the binding activity of the over-expressed protein. Methods:Two pairs of oligonucleotide primers were designed and used to amplify the VH and VL nucleotide sequences,then use VH and VL as template to amplify the scFv. Then the protein gene was cloned into expression vector pBAD-A. The synthesized tP coding sequence was cloned into expression vector pBAD-AscFv,and expressed in E.coli TOP10F′. The fusion protein was detected by SDS-PAGE and Western Blot,then purified by NiNTA chelating agarose. The antigen binding activity was detected by ELISA. And the DNA binding ability was detected by gel shift assay. Results:Restriction digestion and DNA sequencing proved that scFv/tP was correctly cloned into expression vector. SDS-PAGE and Western Blot results showed that scFv/tP fusion protein was successfully expressed in TOP10F′. ELISA detection confirmed that the specific antigen binding activity of the fusion protein; and gel shift assay results suggested its DNA binding ability. Conclusion:The scFv/tP fusion protein successfully expressed in E. coli and could specially bind with both EGFR antigen and DNA.