A thalidomide-binding aptamer was produced by systematic evolution of ligands by exponential enrichment from a library of non-natural DNA in which thymidine had been replaced with a modified deoxyuridine bearing a cationic functional group via a hydrophobic methylene linker at the C5 position. The additional functional group in the modified DNA aptamer could improve stability against nucleases and increase the binding affinity to thalidomide. The selected aptamer could recognize thalidomide enantioselectively, although a racemic thalidomide-attached gel was used for the selection. Surface plasmon resonance and fluorescence titration studies revealed that the selected modified DNA aptamer and a truncated version bound with an (R)-thalidomide derivative with high enantioselectivity, but not with the (S)-form. The modified group in the DNA aptamer is indispensable for the interaction with thalidomide, as the corresponding natural type DNA bearing the same base sequence showed no binding affinity with (R)- nor (S)-thalidomide. Computational sequence analysis suggested that the selected apatamer (108mer) could fold into a three-way junction structure; however, truncation of this aptamer (31mer) revealed that the thalidomide-binding site is a hairpin-bulge region that is a component of one of the arms of the three-way junction structure. The K-d value of the truncated 31mer aptamer for binding with the (R)-thalidomide derivative was 1.0 mu M estimated from fluorescence titration study. The aptamer that can recognize a single enantiomer of thalidomide will be useful as a biochemical tool for the analysis and study of the biological action of thalidomide enantiomers.