With the purpose of developing biosensors, the reliable proof of the biological activity of two new sensor systems was obtained by atomic force microscopy (AFM) in both the imaging and the single-molecule force spectroscopy modes. Antigens or antibodies of pharmacological interest were grafted onto self-assembled monolayers of thiols on gold, and AFM imaging demonstrated that the grafting process produced homogeneous submonolayers of isolated proteins. The analysis of the morphology of the surfaces at the different functionalization steps allowed evaluating the protein grafting density and showed that the recognition of complementary species present in the surrounding solution occurred. Single-molecule force spectroscopy experiments between the sensing surfaces and AFM probes, onto which the complementary species were grafted, enabled a direct and rapid test of the biological activity of the sensors by investigating the interaction occurring at the level of one single ligand-receptor bond. Ellipsometry and surface plasmon resonance allowed further characterization of the sensor surfaces and confirmed that the biological recognition took place.