Based on a green synthesis method, 9 fatty acyl amino acid surfactants were prepared by reacting methyl laurate, methyl myristate and methyl palmitate with alanine, serine and threonine, respectively. The effects of the synergistic interaction of hydrophilic/hydrophobic groups on the interfacial behavior were investigated by surface/interfacial tension measurement and interfacial dilatational rheological experiments. The results showed that, compared with that of alanine, the hydroxyl groups in the α-substituents of the serine and threonine surfactants contributed to the enhancement of intermolecular hydrogen bonding, the improvement of interfacial activity, and the close arrangement of surfactant molecules at the interface. Meanwhile, the increase of fatty acyl chain length made the effect of amino acid α-substituents on the interfacial properties of surfactants more significant. In turn, the polar α-substituent of amino acid facilitated the hydrophilic-lipophilic balance of long-chain fatty acyl surfactants and improved their interfacial properties. Due to the synergistic effect of hydroxyl group and long chain fatty acyl group, the CMC of palmitoyl serinate surfactant declined to 3.9×10_^-4 mol/L, the surface tension of aqueous solution was reduced to 27.60 mN/m, the minimum surface area occupied per surfactant molecule at surface decreased to 0.87 nm₂^{, and the interfacial dilatation}al modulus was enhanced as high as to 47.6 mN/m. Hydroxyl group of amino acid and long fatty acyl group showed a good synergistic effect in enhancing the interfacial properties,.