SARS-CoV-2 has spread worldwide since 2019, causing the devastating COVID-19 pandemic. As of June 23, 2024, the cumulative number of infections worldwide has exceeded 705 million. As the structural protein of SARS-CoV-2, nucleocapsid (N) protein plays a key role in the viral lifecycle and participates in various activities during virus invasion, including interaction with the host immune system. In this study, we used computational methods to predict the MHC-I dominant epitopes, which brought about 282 dominant epitopes restricted to the human HLA-I superfamilies and mouse H-2 haplotypes. Further analysis of immunogenicity and conservation yielded the “preferred epitopes”. Among them, KTFPPTEPK, LSPRWYFYY, SPRWYFYYT and NTASWFTAL have been validated by other laboratories in one or two dimensions in terms of their antigenic profiles. Through our multi-disciplinary research, we also screened out and obtained four unproven “preferred epitopes”. Docking simulations were conducted with the corresponding MHC-I alleles. Then, two-way hierarchical clustering revealed the principles on immunoreacitivities between NP peptides and pan-MHC-I haplotypes. We propose a state-of-the-art epitope exploitation strategy that integrates multiple tools and approaches and provides prospects for the development and application of epitope-based immunotherapy in viral epidemics. Our study also provides insight into extensive protective mechanisms between different variants, providing guidance for the development of highly protected epitope vaccines in the future.