The combination of electron beam lithography and gold nanoparticle-based detection method is subject to a novel high-resolution approach to detecting DNA nanoarrays. In this work, gold nanoparticle-based detection of DNA hybridization on nanostructured arrays is presented. The nanostructured arrays were created by electron beam lithography of a self-assembled monolayer. Amine groups, which are active moieties and are used for attachment of DNA, were introduced to the nanostructures, and the amine-modified structures were characterized by scanning Maxwell-stress microscopy (SMM) for seeing the modification process. The DNA probe covalently immobilized within the nanostructures was hybridized with a biotinylated target DNA. Streptavidin-gold conjugate was then bound to the biotin, thereby assembling inside the nanostructured arrays. The sequence-specific hybridization was imaged by atomic force microscopy (AFM). On the other hand, the activity of the DNA molecules within the nanostructured arrays was verified by fluorescence microscopy using streptavidin-Cy 5 conjugate instead of streptavidin-gold nanoparticles conjugate. On the basis of fluorescent detection, an alternative method has been developed for detection of DNA nanostructures, which will benefit the development of DNA chips.