In this study, a simple and novel electrochemical biosensor based on a glassy carbon electrode (GCE) modified with a composite of graphene oxide (GO) - silk fibroin nanofibers (SF) and gold nanoparticles (MCH/ssDNA/AuNPs/SF/GO/GCE) was developed for detection of DNA sequences. The fabrication processes of electrochemical biosensor were characterized by scanning electron microscopy (SEM), FT-IR and electrochemical methods. Some experimental conditions such as immobilization time of probe DNA and MCH incubation time, time and temperature of hybridization were optimized. The designed biosensor revealed a wide linear range of 1.0 × 10-16 - 1.0 × 10-8 mol L-1 and a low detection limit (3.3 × 10-17 mol L-1) for detection of BRCA1 5382 mutation by EIS technique. The designed biosensor revealed high selectivity for discrimination of the complementary (P1C) sequences from various non-complementary sequences of (P1nC1, P1nC2 and P1nC3). Also, the biosensor revealed a high reproducibility (RSD of 7.5% (n=4)) and high stability (92% of its initial response after 8 days). So, the fabricated biosensor has a suitable potential to be applied for detection of breast cancer sequences in the initial stages of the cancer.