Proteomics has provided a tool to define protein profile of a specific cell or tissue and to associate protein expression levels and post-translational modifications with disease states therefore developing innovative technologies for measurement of protein levels has become a major challenge of the last few years. Specific nucleic acid-based compounds, named aptamers, have been shown as high-affinity ligands and potential antagonists of disease-associated proteins. Aptamers, isolated from combinatorial libraries by an iterative in vitro selection process, discriminate between closely related targets thus representing a valid alternative to antibodies or other bio-mimetic receptors, for the development of biosensors and other bio-analytical methods. Moreover they can be easily stabilized by chemical modifications for in vivo applications and numerous examples have shown that stabilized aptamers against extracellular targets such as growth factors, receptors, hormones or coagulation factors are very effective inhibitors of the corresponding protein function. By integrating the aptamer-based biosensor development with the maturing technology for in vitro selection of anti-protein aptamers results in the highthroughput production of proteome chips. Furthermore, aptamer arrays and biosensors will reveal the most effective tools for the detection of biomolecular interactions and the identification of protein targets, particularly with regard to those not detectable by known receptors like enzymes or antibodies. We will review here the main and innovative methods based on the use of aptamers as biosensors for protein detection that, in alternative or combined to the classical proteomic approaches, could reveal suitable for both diagnostic and therapeutic purposes.