The present study investigated the mechanism of the chiral recognition of the resolving agent (L-phenylalanine) to the chiral isomers (D/L-mandelic acid). According the NMR analysis, the distinctive chemical shifts of between two diastereomer crystals (L-mandelic acid-L-phenlyalanine and D-mandelic acid-L-phenylalanine) were observed even though there was no difference of the chemicals shift of the two diastereomer solutions. This result indicated that the chiral recognition of the resolving agent mainly occurred during the crystallization of the diastereomers in the solution. Then, the chiral recognition of the diastereomers was confirmed by using thermal analysis and AFM. The diastereomer crystal of L-mandelic acid-L-phenylalanine was much more thermally stable due to the higher lattice energy than the diastereomer crystals of D-mamdelic acid-L-phenylalanine. Also, the adhesive force measured with AFM exhibited a stronger molecular interaction between L-mandelic acid and 4-amino-L-phenylalanine than between D-mandelic acid and 4-amino-L-phenylalanine. Plus, the AFM results implied that the hydroxyl group abundance on the mandelic acid surface was a possible explanation for the different chiral selectivity of the L-phenylalanine.