The development of broad-spectrum universal influenza vaccines and optimisation of vaccination strategies to address the threats posed by pandemics and emerging influenza viruses are critical for public health. In this study, an adenovirus type 5 vector-based influenza vaccine carrying the hemagglutinin (HA) stem of H1, HA stem of H3, and neuraminidase of N1 from the influenza virus was constructed. Immune responses were evaluated in mice using various vaccination strategies: prime-only (intramuscular [IM] or intranasal [IN]) and prime-boost (IM+IN). Compared to the prime-only strategy, the prime-boost strategy significantly enhanced the systemic immune response, inducing higher levels of antigen-specific IgG, mucosal IgA, and T cell immunity in the spleen and lungs. Furthermore, the IN boosting strategy provided complete protection in mice challenged with the H1N1-PR8, rgH3N2-X31, and rgH5N1-Vietnam viruses, significantly reducing viral loads in the lungs and alleviating lung tissue pathologies. In conclusion, this study elucidates potential avenues for the development and application of universal influenza vaccines using customised mucosal boosting strategies.