Nanoelectrode ensembles (NEEs) obtained by electroless gold deposition in track-etched poly-carbonate (PC) membranes are functionalized and applied for DNA hybridization detection, using methylene blue (MB) as electroactive probe. To this aim, an amine terminated (ss)DNA probe is immobilized on the PC surface of the NEE by reaction via carbodiimide and N-hydroxysulfosuccinimide. In order to increase the number of carboxylic groups present on PC and suitable for the functionalization, the surface of NEEs is oxidized with potassium permanganate. The presence of carboxylic functionalities is verified by spectrochemical titration with thionin acetate (THA) and the effect of the activation treatment on the electrode performances is evaluated by cyclic voltammetry (CV). After activation and functionalization with the probes, the NEE-based sensor is hybridized with complementary target sequences. The effect of the functionalization of the NEEs both with the (ss)DNA probe alone and after hybridization with the target, is studied by measuring the changes in the MB reduction signal by square wave voltammetry (SWV), after incubation in a suitable MB solution, rinsing and transfer to the measurement cell. It was observed that this peak signal decreases significantly after hybridization of the probe with the complementary target. Experimental evidences suggest that the interaction between MB and the guanines of (ss)DNA and (ds)DNA is at the basis of the development of the here observed analytical signal. The proposed approach allows the easy preparation and testing of NEE-based sensors for the electrochemical DNA hybridization detection.