Nucleic acid aptamers are short synthetic DNA or RNA sequences which can bind to a wide range of targets with high affinity and specificity. In recent years, aptamers have aroused increasing research interest due to their unique features of high binding affinity and specificity, small size, excellent chemical stability, easy chemical synthesis, facile modification, and minimal immunogenicity. These properties make aptamers ideal targeting ligands for bioanalysis, disease diagnosis and cancer therapy. This review highlights recent progress in aptamer selection and the latest applications of aptamer-based functional probes in the fields of bioanalysis and biomedicine.