Melittin (MLT) is a small cationic peptide discovered from the bee venom. It is used as an antimicrobial agent due to its broad-spectrum activities against bacteria, fungi and tumor cells. But the sources limit its applications. Therefore, the small ubiquitin-related modifier (SUMO) fusion technology was reported for high-level expression of Melittin. pET-3c-SUMO-Melittin plasmid was constructed, and the fusion protein (SUMO-Melittin) was expressed in a soluble form and purity by Ni2+-NTA chromatography. After the SUMO-Melittin fusion protein was cleaved by the SUMO protease, the cleaved sample was purified again by a Ni2+-NTA. Finally, about 25 mg recombinant Melittin was obtained from 1L fermentation culture with more than 95% purity. The recombinant Melittin exhibited similar cytotoxicity and antimicrobial properties as the synthetic Melittin. Thus, a system for high-level expression of Melittin was successfully established in this study and could be used for preparation of similar antimicrobial peptides.