DNA-programmed nanomaterials represent a revolutionary convergence of nanotechnology and molecular biology, offering unprecedented precision in the design and application of functional nanostructures. By leveraging the programmability of DNA base-pairing, molecular recognition, and inherent biocompatibility, researchers have developed diverse DNA-engineered nanomaterials for cutting-edge applications in biosensing, bioimaging, and therapeutic delivery. In this review, we systematically explore the construction and functionalization of DNA-conjugated nanomaterials (e.g., DNA-gold nanoparticles, DNA-upconversion nanoparticles, DNA-metal-organic frameworks) and DNA-templated assemblies (e.g., metal nanoclusters, quantum dots), highlighting their tailored physicochemical properties and dynamic responsiveness. Furthermore, we discuss their critical roles in early disease diagnosis, real-time molecular imaging, and precision medicine. By providing a comprehensive overview of recent advancements, this review aims to enhance understanding of the current landscape and inspire future innovations in the controllable assembly and biomedical applications of DNA-programmed nanomaterials.