Chemical modifications of innate DNA/RNA aptamers facilitate the improvement of their function. Herein, we report our modular strategy to manipulate a thrombin-binding DNA aptamer (TBA) to improve its anticoagulation activity and binding affinity. A set of amino acid conjugates, termed amino acid-nucleic acid hybrids or ANHs, was synthesized and incorporated into a TBA loop sequences. We found that substitutions with hydrophobic amino acids in the loop region possessed significantly enhanced antithrombin activity, up to 3-fold higher than the native TBA. We investigated the correlations between thrombin-binding affinity and the features of our amino-acid conjugates using experimental techniques including circular dichroism spectroscopy, surface plasmon resonance assay, and molecular modeling. The present study demonstrates a systematic approach to aptamer design based on amino-acid characteristics, allowing the development of advanced aptamers.