Chiral recognition at single-molecule level for small active molecules is important, as exhibited by many nanostructures and molecular assemblies in biological systems, but it presents a significant challenge. We report a simple and rapid sensing strategy to discriminate all enantiomers of natural aromatic amino acids (AAA) using a metal–organic complex-functionalized protein nanopore, in which a chiral recognition element and a chiral recognition valve were equipped. A trifunctional molecule, heptakis-(6-deoxy-6-amino)-β-cyclodextrin (am₇βCD), was non-covalently lodged within the nanopore of an α-hemolysin (αHL) mutant, (M113R)₇-αHL. Copper(II) ion reversibly bonds to the amino group of am₇βCD to form an am₇βCD-Cu^II complex, which allowed chiral recognition for each enantiomer in the mixture of AAA by distinct current signals. The Cu^II plugging valve plays a crucial rule that holds chiral molecules in the nanocavity for a sufficient registering time. Importantly, six enantiomers of all nature AAA could be simultaneously recognized at one time. Enantiomeric excess (ee) could also be accurately detected by this approach. It should be possible to generalize this approach for sensing of other chiral molecules.