DNA is a powerful biomaterial for creating rationally designed and functionally enhanced nanostructures. DNA nanoarchitectures positioned at substrate interfaces can offer unique advantages leading to improved surface properties relevant to biosensing, nanotechnology, materials science, and cell biology. This Perspective highlights the benefits and challenges of using assembled DNA as a nanoscale building block for interfacial layers and surveys their applications in three areas: homogeneous dense surface coatings, bottom-up nanopatterning, and 3D nanoparticle lattices. Possible future research developments are discussed at the end of the Perspective.