The self-assembly behavior of OA-amino acid conjugates was systematically investigated, providing insight into how slight structural modifications could alter the self-assembly behaviors. The conjugates OA-Gly-OH, OA-Thr-OH, and OA-Phe-OH exhibited distinct CAC as determined using pyrene fluorescence. The role of amino acid side chain properties in altering hydrophilicity, with glycine showing the highest hydrophilicity and phenylalanine the lowest. This variation directly impacted the selfassembly behavior, as evidenced by the significantly different CAC values. TEM imaging further revealed difference in the resulting self-assembly structures, demonstrating the versatility of these conjugates in forming supramolecular structures. These findings underline the potential of fatty acid-amino acid conjugates as tunable amphiphiles for diverse applications in drug delivery, biomedicine and materials science.