Hollow polymer microspheres (HPMs) were synthesized, which were then hydrolyzed in aqueous ammonia to produce carboxyl (–COOH) groups on their surface. L-phenylalanine (L-Phe) was grafted to the hydrolyzed HPMs (H-HPMs) through amidation reactions, endowing the H-HPMs with chirality. The resultant chiral HPMs (C-HPMs) were used for the chiral discrimination of tryptophan (Trp) isomers. Due to the same rotatorydirection of L-Phe and L-Trp, the C-HPMs showed greatly higher selectivity toward L-Trp than its isomer. After being adsorbed by the C-HPMs, the absorbance of the residual L-Trp is significantly lower than that of the residual D-Trp, and thus spectroscopic chiral discrimination of the Trp isomers was successfully achieved. The Trp isomers were also discriminated by the chiral solid polymer microspheres (C-SPMs), while the difference in the absorbance of the residual L-Trp and D-Trp is remarkably smaller than that obtained by the C-HPMs. The outstanding discrimination capability of the C-HPMs might be ascribed to their high surface permeability resulted from their unique hollow structure.