Objective:To construct E.coli expression vector of human spasmolytic polypeptide(hSP),express recombinant hSP and underlie the base of function analyses. Methods:The hSP gene encoding mature peptide was amplified by reverse transcriptase polymerase chain reaction,and then inserted into the expression vector pET32a. Recombinant plasmid pET32a-hSP was transformed into the E.coli Origami B(DE3). Recombinant proteins were assayed with SDS-PAGE and Western blot. Results:It was proved that the fragment amplified was inserted into the expression vector pET32a correctly by PCR and gene sequencing. Tricine SDS-PAGE analyse proved that the molecular weight of hITF was about 32 kD and Western blot demonstrated that the expression proteins have much good antigenicity and specificity. Conclusion:hSP E.coli expression vector was successfully constructed and recombinant hSP was expressed. This research lay foundations for the further function studying of hSP.