BACKGROUND: The clinical application of peptide vaccines in tumor immunotherapy holds significant promise. Peptide-based tumor vaccines are currently subject to certain limitations in clinical trials, including the challenge of inducing a sustained response from CD4 T helper cells and cytotoxic T lymphocytes (CTL), as well as human leukocyte antigen (HLA) restrictions. METHODS: Through the utilization of biological information methodology, a screening process was conducted to identify three potential long peptides that are specifically targeted by the MAGE-A4 antigen. The candidate long peptides were subjected to in vitro testing using human peripheral blood lymphocytes as samples to evaluate their immunogenicity and immune function. The antitumor properties and preliminary mechanism of the long peptide vaccine were investigated through the use of a mouse model designed for the prevention of triple negative breast cancer (TNBC). RESULTS: Three predicted multi-epitope long peptides targeting MAGE-A4 have shown to have a strong immunogenicity, with a total positive rate of 72% across different HLA subtypes in Chinese populations. they can also increase the levels of the costimulatory factor CD137 and tumor necrosis factor-alpha (TNF-α), activate T cells, and boost the cytotoxic activity. Results from an animal study have revealed that the long-peptide vaccine, both on its own and in combination with R848, has displayed impressive anti-tumor and target-specific capabilities. Moreover, it has the ability to increase the expression of effector memory T cells and central memory T cells. CONCLUSIONS: This study was the first to screen three multi-epitope long peptides targeting MAGE-A4 and assess their immunogenicity, immune function, and potential as adjuvant peptides. The results showed that the MAGE-A4 long peptide vaccine can be used as a novel immunoprophylaxis method to prevent TNBC. Moreover, the proposed development model is capable of screening multiple target antigens, which lead to its clinical application.