Hydrothermally prepared mono-6-SH-β-cyclodextrin capped Mn-doped ZnS quantum dots (β-CD-Mn-ZnS QDs) exhibited dual photoluminescence (PL) at 430 nm and 598 nm, respectively, upon excitation at 315 nm. The PL intensity of β-CD-Mn-ZnS QDs responded to tryptophan enantiomers differently: the D-isomer showed little effect while L-tryptophan displayed a large time-dependent enhancement in the PL intensity of QDs. This chiral selectivity originated from the β-cyclodextrin coating on the surface of Mn-ZnS QDs, which shows different inclusion constants for tryptophan enantiomers. β-CD-Mn-ZnS QDs further selectively hydrolyse L-tryptophan, forming a product that greatly enhances the QD photoluminescence. Based on these findings, a photoluminescence chiral-assay for tryptophan enantiomers was developed. L-Tryptophan can be detected in the presence of its stereoisomer with a detection limit of 5.4 nM in a linear range of 0–6.0 μM.