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Clinical diagnosis has always been dependent on the efficient immobilization of biomolecules in solid matrices with preserved activity, but significant developments have taken place in recent years with the increasing control of molecular architecture in organized films. Of particular importance is the synergy achieved with distinct materials such as nanoparticles, antibodies, enzymes, and other nanostructures, forming structures organized on the nanoscale. In this review, emphasis will be placed on nanomaterials for biosensing based on molecular recognition, where the recognition element may be an enzyme, DNA, RNA, catalytic antibody, aptamer, and labeled biomolecule. All of these elements may be assembled in nanostructured films, whose layer-by-layer nature is essential for combining different properties in the same device. Sensing can be done with a number of optical, electrical, and electrochemical methods, which may also rely on nanostructures for enhanced performance, as is the case of reporting nanoparticles in bioelectronics devices. The successful design of such devices requires investigation of interface properties of functionalized surfaces, for which a variety of experimental and theoretical methods have been used. Because diagnosis involves the acquisition of large amounts of data, statistical and computational methods are now in widespread use, and one may envisage an integrated expert system where information from different sources may be mined to generate the diagnostics