Although most in vivo biomolecular recognition occurs in solution, in many practical situations (e.g., diagnostics, drug discovery and biosensing) biomolecular recognition occurs between “target” biomolecules immobilised on surfaces and “probe” complementary biomolecules approaching the surface from solution. DNA-based devices are by far the most common biomolecular and cellular planar biodevices with a still-commanding growth rate. A second, but chronologically older, interest derives from the need to understand the fundamentals of biomolecular interactions at single molecule level and in large supramolecular assemblies. Again, DNA molecules are not only essential objects of study, but also more attractive candidates as the building blocks of artificial biomolecular devices than, e.g., proteins, because of their relative simplicity and robustness. Among the many microscopy-based techniques for the study of biomolecular interactions on surfaces, scanning probe microscopies, and especially the atomic force microscopies (AFM), are the most used because of their molecular and sub-molecular level resolution and in situ imaging capability. Apart from the high resolution mapping of surface nanotopographies, AFM can be used for the quantification and visualisation of the distribution of chemistry, hydrophobicity and local mechanical properties on surfaces, and for the fabrication of nanostructures on surfaces. The present article, which reviews from classical and latest developments regarding AFM studies of DNA molecules immobilised on surfaces, is organised along the nature of DNA aggregates on surfaces, i.e., single molecules, self-assembled layers and amorphous layers, with the last two emerging areas receiving a relatively higher emphasis. Within these three areas of application, the material is organised along the main functions of the AFM, namely imaging, probing biomolecular interactions and fabrication of nanodevices.