Abstract: To construct tir-stx fusion gene, the DNA fragments encoding middle 295 amino acids （Tir295）of tir and 72 amino acids （Stx72）of Stxlb and Stx2b were fused in turn in frame with the multiple cloning sites of pET-28a, and the recombinant plasmid of pET28a- Stx2b-Tir-Stxlb was transferred to host cells E.coli BL21 strain （DE3）. Then, the protein was induced and expressed with induction of IPTG. The expression quantities and style of fusion protein were determined by SDS-PAGE and thin layer scanning results showed that amount of expressed fusion protein was 30% in total of bacteria protein. Because the fusion protein was consisted of tir295, and stxb72 antigens, it can evoke immune responses against tir295 and stxb72. The tir-stxb fusion protein expressed in E.coli could offer an important ingredient that should be consider in construction of recombinant EHEC O157 vaccine or preparation of experimental material for the monoclonal antibody.