The innate immune system is the first line of the multicellular organism against the various pathogens invading. The Notch signaling plays a significant role in maintaining the balance among cell proliferation, differentiation, apoptosis and development. To further investigate the function of Notch signaling pathway in Drosophila innate immunity, Drosophila mutants, E(spl) and Su(H), which are located in the downstream of Notch signaling pathway, were utilized to analyze the survival rate, in vivo phagocytosis and production of antimicrobial peptides after pathogen infections. In addition, the number of circulating plasmatocytes in third instar larvae of mutant and wild type flies was also measured. The results indicate that the E(spl) mutant showed lower survival rate, phagocytosis and the expression of AMP genes after pathogen infections. Furthermore, an abnormal increase in the number of plasmatocytes in E(spl) mutant larvae was also observed. Compared with E(spl) mutant, the mutant of Su(H) only showed sensitive to fungi, however, phagocytic ability of hemocytes was normal in fungi infection. At the same time, the expression of antimicrobial peptides was decreased in Su(H) mutant. These results show that the Notch pathway not only affects the growth and development of individuals, but also plays an important role in regulating innate immunity of Drosophila.