Aptamers are small nucleic acid molecules capable of binding to a wide range of target molecules with high affinity and specificity. They have been developed and widely used not only as research tools, but also as biosensors, specific antagonists, and diagnostic markers and as protein purification platform for many pharmaceutical and clinical applications. Here, in this paper we will explore biochemical aspects of aptamer–target interactions and show why aptamers rival antibodies in target recognition and purification procedures. This review will focus on strategies of using aptamers as affinity ligands for molecules of therapeutic and pharmaceutical interest including applications in chromatography and capillary electrophoresis for protein and small molecule purification. Moreover, we will also discuss aptamers whose binding parameters can be controlled on demand for diagnostic approaches and used as sensitive receptors in biosensorics. Aptamers have opened up exciting fields in basic and applied research of pharmaceutical and biotechnological interest.