Background The frequent outbreaks of respiratory viral infections pose a severe threat to human health. Broadspectrum antiviral agents such as interferons may serve as emergency prophylactic measures during the early stages of viral pandemics and epidemics, yet existing studies lack systematic efficacy evaluations.Objective To investigate whether intranasal administration of interferon α2 effectively activates respiratory immune responses, and evaluate the potential role of interferon α2 in preventing respiratory viral infections using influenza A (H1N1) virus infection model.Methods Mice were intranasally pre-treated with interferon α2 according to different administration schedules and dosage gradients. The changes in lung interferon responses were monitored to determine optimal administration conditions. Mice were subjected to preventive treatment under these conditions, followed by analyses of cytokine expression profiles in lungs, and infection with the influenza A (H1N1) virus to assess the protective efficacy of interferon α2.Results The results indicated that intranasal administration of interferon α2 induced dose- and time-dependent interferon responses in the lungs, and the peak was attained two hours after administering 2×105 U of interferon α2. Employing this administration regimen for a single-dose intranasal preventive treatment significantly upregulated antiviral cytokines including IL-28 in lungs (P < 0.05), and effectively reduced viral titers in upper respiratory tracts (P < 0.05), ameliorated pathological damage such as pulmonary edema and hemorrhage (P < 0.05) and extended survival in mice challenged with a lethal dose of influenza A (H1N1) virus (P < 0.01).Conclusion This study demonstrates that intranasal administration of interferon α2 induces an effective interferon response in the lungs, providing protection against influenza A (H1N1) virus infection, which lays a foundation for the application of interferons in the prevention and treatment of respiratory viral infections.