A novel nanostructured electrode material based on electrochemically reduced graphene oxide/polyaniline nanowires/silver nanoflowers (ERGO/PANi NWs/AgNFs) was fabricated site-specifically onto a Pt microelectrode (0.80 mm² area) using a three-step electrochemical procedure: electrosynthesis of ERGO, electropolymerization of PANi NWs, and electrodeposition of AgNFs. Synergistic and complementary properties of ERGO, PANi NWs and AgNFs, including high electrochemical activity, large surface area, and high biocompatibility, were obtained. Besides, the electrosynthesis method allowed the direct formation of the desired nanomaterial onto the Pt microelectrode, so the adhesion between the sandwich-structured nanocomposite and the electrode surface was also improved. The optimized ERGO/PANi NWs/AgNFs nanocomposite was used for the first time to develop an electrochemical DNA sensor. As a result, the DNA probe immobilization was facilitated and the electrochemical signals of the DNA sensor were enhanced. The detection limit of the DNA sensor was 2.70 × 10⁻¹⁵ M. Moreover, potential miniaturization for fabrication of a lab-on-a-chip system, direct detection, high sensitivity, and good specificity are the advantages of the fabricated DNA sensor.