In addition to its role as a carrier of genetic information, DNA has been recognized as a construction material for the assembly of different objects and structural arrangements with nanoscale features. As a result of DNA’s self-recognition properties (based on the specific base-pairing of G-C and T-A), monolayer films of nucleic acids on solid supports have attracted an escalating attentions. Recently, numerous novel materials based on two-dimensional (2D) and three-dimensional (3D) DNA structures have been reported, which extends their utility to a large number of appliations. This review paper intends to be a new and comprehensive overview of recent strategies to site-specifically immobilized DNA on various materials, including carbonaceous substances, gold, and silica substrate, emphasizing the applications of site-specific DNA nanostructure-based devices for diagnostic, bioanalytical, food safety and environmental monitoring. Additionally, an up-to-date perspective is proposed at the end of this review.