Cancer is one of the leading causes of death worldwide and a crisis for global health. Breast cancer is the second most common cancer globally. In the perusal, a novel electrochemical biosensor amplified with hierarchical flower-like gold, poly (n-butyl acrylate), and MXene (AuHFGNs/PnBA-MXene) nanocomposite and activated by highly special antisense ssDNA (single-stranded DNA) provide a promising alternative for miRNA-122 detection as a biomarker of breast cancer. The biosensor presented a detection limit of 0.0035 aM (S/N = 3) with a linear range from 0.01 aM to 10 nM. The platform was tried on 20 breast cancer miRNAs extracted from actual serum specimens (10 positives and 10 negatives). Founded on the quantitatively obtained outcomes and statistic analysis (t-test, box-graph, receiver performance characteristic curve, and cut-off amount), the biosensor showed a meaningful discrepancy between the native and positive groups with 100% specificity and 100% sensitivity. While, RT-qPCR showed less specificity and sensitivity (70% specificity, 100% sensitivity) than the proposed biosensor. To assess the quantitative capacity and biosensor detection limit for clinical tests, the biosensor diagnosis performance for continually diluted miRNA extracted from patients was compared to that gained by RT-qPCR results, indicating that the biosensor detection limit was lower than RT-qPCR. ssDNA/AuHFGN/PnBA-MXene/GCE displayed little cross-reaction with other sequences and also showed desirable stability, reproducibility, and specificity and stayed stable until 32 days. As a result, the designed biosensor can perform as a hopeful method for diagnosis applications.