Nucleic acid aptamers isolated from the systematic evolution of ligands by exponential enrichment (SELEX) have the capacity to recognize various classes of target molecules with high affinity and specificity. In this context, the development of aptamer-based molecular recognition tools has become a very interesting and promising analytical strategy during the last few years. In this review, the molecular recognition features of aptamers as well as the key factors for their practical applicability to the chromatographic and capillary electrophoretic fields are summarized.