Object: To study Expression and Purification of antimicrobial Peptide S-thanatin in Escherichia coli and the comparison of different MIC methods about S-thanatin. Methods: In this study, a recombinant plasmid pET32a-Ts coding for fusion protein in Escherichia coli was constructed by recombinant gene technique. The fusion protein was purified through Nickel-affinity chromatography column, then cut off TRX with thrombin after dialysis, purified by Sephadex G-50 chromatography, and finally freeze-dried. Constant broth dilution method, microdilution method and punching method were used to observe the MIC of S-thanatin. Results: S-thanatin was successfully expressed in E.coli, and the final purity was higher than 85%. Different methods of MIC about Ts showed different results, the constant broth dilution method and PP 96 pores plate gave the best effect. Conclusion：Ts was highly expressed in engineering strain BL21(DE3), and the purified protein had a high biological activity. MIC of PP 96 and constant broth dilution method gave the accurate result. The antibacterial activity was affected by the experimental material.