A gold-nanostructured surface has been obtained by stable deposition of chemically synthesized gold nanoparticles (2.1–5.5 nm size range) on a gold substrate through a dithiol linker. The method proposed for the obtainment of the nanostructure is suitable for the further stable anchoring of a peptide nucleic acid oligomer through four amine groups of lysine terminal residues, leading to fairly reproducible systems. The geometric area of the nanostructured surface is compared with those of a smooth and of an electrochemically generated nanostructured surface by depositing a probe bearing an electrochemically active ferrocene residue. Despite the area of the two nanostructures being quite similar, the response toward a 2 nM target oligonucleotide sequence is particularly high when using the surface built up by nanoparticle deposition. This aspect indicates that morphologic details of the nanostructure play a key role in conditioning the performances of the genosensors.