Abstract: Musca domestica Phormicin, belonging to the defensin super family, is a kind of antimicrobial peptides with broad antibacterial activity. In this study, Phormicin A protein of housefly was expressed and purified by prokaryotic expression method. And it was mixed and emulsified well with complete and incomplete adjuvants. Then the polyclonal antibodies were obtained after immunizing New Zealand white rabbits. The specificity of the antibody was verified by neutralization and adsorption of prokaryotic expression protein incubated with antibody via Western blot experiments. Endogenous validation was performed and the antibody titer was also determined by using the samples of 3 rd instars larvae of housefly stimulated by Staphylococcus aureus. The results showed that the polyclonal antibody could recognize not only the prokaryotic expression protein, but also the endogenous Phormicin A protein produced from 3 rd instars larvae of housefly stimulated by the S. aureus. This study laid a foundation for further exploration of the mechanism of Phormicin in innate immunity and defense of housefly.