In this work, we synthesized novel polymer dots (Pdots) from polymers of intrinsic microporosity (PIM-1) containing a racemic mixture of R- and S-chiral centers as the hydrophobic conjugated polymer chains. Although PIM-1 showed no enantiomeric recognition abilities toward amino acids, we found that this ability can emerge after converting these hydrophobic polymers into nanosized water-soluble Pdots, using poly(styrene-co-maleic anhydride) (PSMA) as a co-polymer that provides carboxyl functional groups on the surface of the Pdots. The stronger interaction of Pdots with amino acids and their higher ability to discriminate the amino acid enantiomers were clearly confirmed by the quantum mechanical calculations, where the difference between the calculated energies for the interaction of D- and L-amino acid enantiomers with the simulated Pdots were remarkably higher than those obtained for the interaction of the enantiomers with just PIM-1. Indeed, the carboxyl surface groups of Pdots played a critical hook-like role in bringing the amino acid enantiomers and chiral centers of the embedded PIM-1 closer together to provide more efficient interactions. The synthesized Pdots were successfully used as highly efficient chiral probes for chiral recognition and determination of the enantiomers of (Phe), tryptophan (Trp), and tyrosine (Tyr) as samples by both fluorescence and circular dichroism spectroscopic methods. The results of this study demonstrated the potential of the suggested method to utilize hydrophobic chiroptical probes in enantiomeric recognition of hydrophilic chiral species.