Developing an effective universal influenza vaccine against influenza virus with highly conserved antigenic epitopes could induce a broad-spectrum immune response to prevent infection. The soluble protein M1 that can induce the M1 specific immune response was first confirmed in our previous study. In this study, we characterized the immune response induced by DNA prime-subunit protein boost strategy based on the relatively conserved matrix protein 1 (M1) in the BALB/c mouse model, and evaluated its protection ability against a lethal challenge of homologous H9N2 avian influenza virus (A/Chicken/Jiangsu/11/2002). The results showed that 100 μg DNA prime + 100 μg M1 subunit protein boost-strategy significantly increased antibody levels more than vaccination with M1 DNA or M1 subunit protein alone, and induced a more balanced Th1 / Th2 immune response, which not only can provide protection against the homologous virus but also can provide part of the cross-protection against the heterosubtypic PR8 H1N1 strain. In addition, we used an Elispot assay to preliminary screen the T cell epitope in M1 protein, and identified that p22 (M111-25 VLSIIPSGPLKAEIA) epitope was the only immunodominant M1-specific CD4+ T cell epitopes, which could be helpful in understanding the function of influenza virus T cell epitopes.