Abstract Background Globally, breast cancer is the most frequently diagnosed malignancy and the second leading cause of cancer-related mortality among women. The ongoing pursuit of early detection has driven interest in identifying and validating novel diagnostic biomarkers that could enhance prognosis and therapeutic outcomes. Humanin, a mitochondrial-derived peptide (MDP) with reported cytoprotective properties, has been implicated in cancer biology and may play a role in breast cancer pathogenesis. Methods A total of 75 female patients with primary non-metastatic breast cancer and 70 age-matched healthy controls of comparable age were enrolled in this study. Serum concentrations of humanin were quantified using an enzyme-linked immunosorbent assay (ELISA). Results Serum humanin concentrations were significantly elevated in breast cancer patients compared with healthy controls (p < 0.001). Receiver operating characteristic (ROC) curve analysis demonstrated that humanin effectively differentiated breast cancer patients from controls, with a sensitivity of 78.7% and specificity of 62.9%. Conclusion These findings suggest that humanin could serve as a promising biomarker for breast cancer screening and early detection. Further large-scale studies are warranted to validate its diagnostic potential and establish its clinical utility. Keywords Breast cancer, Humanin, Mitochondrial-derived peptides, Biomarker