MicroRNAs (miRNAs) are small, endogenous, noncoding RNAs, shown to be expressed abnormally in many tumors and identified as predictive biomarkers for early diagnosis of several cancers including the breast. Therefore, the label-free and highly sensitive detection of miRNAs is of critical significance. In this work, a highly sensitive and label-free nano-genosensor is developed for the detection of miRNA-21, a known breast cancer biomarker, based on a specific architecture of nitrogen-doped functionalized graphene (NFG), silver nanoparticles (AgNPs), and polyaniline (PANI) that resulted in a remarkable effect on signal amplification. Following the successful functionalization of the nanocomposite and immobilization of the specific sequence of the aminated complementary oligonucleotide of miRNA-21, the detection was performed using differential pulse voltammetry (DPV). The oxidation peak current of the redox probe under optimal conditions was determined to monitor the event hybridization of miRNA-21 biomarker. Applying this highly sensitive and optimized nano-biosensor enabled detection of a wide dynamic range of 10 fM–10 µM with a sensitivity of 2.5 µA cm⁻² and a low detection limit of 0.2 fM. This nano-biosensor also demonstrated highly reproducible results in the analysis of blood samples, with recoveries between 94% and 107%, and could be used for early detection of breast cancer by direct detection of the miRNA-21 in real clinical samples without any need to sample preparation, RNA extraction and/or amplification.