A novel aptamer generation method to greatly augment the affinity and stability of DNA aptamers was developed by genetic alphabet expansion combined with mini-hairpin DNA technology. The genetic alphabet expansion increases the physicochemical and structural diversities of DNA aptamers by introducing extra components, unnatural bases, as a fifth base, allowing for the enhancement of DNA aptamer affinities. Furthermore, the mini-hairpin DNA technology stabilizes DNA aptamers against nuclease digestion and thermal denaturation, by introducing an extraordinarily stable mini-hairpin DNA containing a GCGAAGC sequence. This novel method provides stabilized high-affinity DNA aptamers for diagnostic and therapeutic applications.