Aptamers are small single-stranded nucleic acids that fold into a well-defined three-dimensional structure. They show a high affinity and specificity for their target molecules and inhibit their biological functions. Aptamers belong to the nucleic acids family and can be synthesized by chemical or enzymatic procedures, or a combination of the two. They can, therefore, be considered as both chemical and biological substances. This Review summarizes the most convenient approaches to their preparation and new developments in the field of aptamers. The application of aptamers in chemical biology is also discussed.