The ability of holding back the undesired molecules, but at the same time to provide the right distribution and orientation of the bioreceptors, are critical targets to reach an efficient hybridization and enhanced detection in electrochemical DNA biosensors. The main actors responsible of these key functions are the substrate of the sensor and the interface auto‐assembled on it. In this paper we present the annealing as a method to improve commercial gold evaporated substrates for biosensor applications. The restructuring of granulated gold surface by means of annealing heating treatment leads to the formation of ultraflat gold lamellar terraces. The formation of terraces was characterized with scanning tunneling microscopy and optical interferometry. The performance of the sensor sensitivity on granular substrates and ultraflat substrates was studied, concerning the orientation and surface coverage of the bioreceptor interface applied in electrochemical biosensor. The hybridization efficiency of ferrocene‐labeled DNA amplified by PCR was characterized with surface plasmon resonance and electrochemistry. The experimental results demonstrate that annealing process, positive influence on optical and voltammetric readings, due to a structured organization of the bioreceptors on the flat substrate, gaining more efficient immobilization and DNA hybridization. The results suggest the annealing as a powerful tool for improving gold substrates in biosensors applications.