The new sensitive diagnostic method, electrochemical nanobiosensor, is applied to cancer detection by using specific biomarkers such as miRNA. Studies have shown that for the diagnosis of Triple Negative Breast Cancer (TNBC) patients, in the early stages of the disease, miR-199a-5p as circulating miRNA can be a suitable candidate. In this study, a new electrochemical nanobiosensor for serum miR-199a-5p detection was designed by using modified glassy carbon electrode (GCE) with graphene oxide (GO) and gold nanoroad (GNR) on which a thiolated probe was immobilized. The electrochemical impedance method (EIS) was used to examine the electrochemical characteristics and behavior of nanobiosensor and for verifying the nanomaterial synthesis the scanning electron microscope (SEM) were applied, field emission scanning electron microscope (FE-SEM), UV–Vis spectrophotometry and energy dispersive spectroscopy (EDS). Optimum conditions were investigated for designed nanobiosensor and in optimal conditions, following results were obtained; the linear range of the calibration curve from 15 fM to 148 pM, the detection limit of 4.5 fM, and the standard deviation of 2.9%. The research data showed that our designed electrochemical nanobiosensor has a sensitivity and desirable precision for evaluation of miR-199a-5p that can be used to measure low concentrations of miR-199a-5p in blood serum sample.