Molecular recognition between specific receptors and ligands is a key step in diverse biological processes and forms the basis of many biosensors. These may include enzyme-substrate, antigen-antibody, lectin-carbohydrate binding. Force spectroscopy using an atomic force microscope (AFM) is a valuable tool for studying and detecting such interactions and molecular biorecognition at a single molecule level. The common practice is to apply a small concentration of protein to a surface and to use statistical techniques to infer the expected characteristics of the interaction between single molecule pairs. Whether the surface-bound proteins are indeed single and isolated remains unclear and often undesirable non-specific protein/surface interactions obscures informative features of the interaction. In this study, mixed self-assembled monolayers (SAMs) consisting of N-hydroxysuccinimide (NHS) and oligoethylene glycol (OEG)-terminated thiols on ultraflat gold surfaces were used to covalently immobilize proteins via lysine residues. By the optimization of attachment sites via lysine-NHS linkages on a protein-resistant layer of the OEG SAM, it is possible to isolate single proteins for study in a controlled fashion. Single protein distribution on the surface is clearly demonstrated by AFM imaging. The OEG-thiol also significantly reduces non-specific tip-surface interactions between the AFM cantilever and surface. The utility of this self-assembled monolayer platform is demonstrated in studies of the interactions of carbohydrate binding proteins (lectins) with sugars. The specific interaction profiles and rupture forces for lectin-carbohydrate binding under physiological conditions will be shown. This platform provides an attractive strategy to fabricate single molecule sensors and label free detection devices.