The stable integration of a biological recognition element on a transducing substrate surface is the single most important step in the creation of a high-functioning sensor surface. The key factors affecting biotic and abiotic functionalities at the biointerface are both chemical and physical. Understanding the interactions between biomolecules and surfaces, and their emergent complexity, is critical for biointerface implementation for sensing applications. In this overview, we highlight materials and methods typically used for biosensor development. Particular emphasis has been given to the experimental evaluation of biointerfacial properties and functionality. Promising research directions for application of biointerfaces to biosensing are suggested.