β-Cyclodextrin (β-CD) modified silver nanoparticles (AgNPs), denoted as β-CD/AgNPs, were prepared by a simple one-pot method. Due to the inherent chirality of β-CD, the developed β-CD/AgNPs exhibited higher affinity toward l-tyrosine (l-Tyr) than d-tyrosine (d-Tyr), leading to serious aggregation of AgNPs in the presence of l-Tyr. Consequently, the l-Tyr induced aggregation of AgNPs can result in signal amplification in the differential pulse voltammograms (DPVs) of l-Tyr, which can be applied for the electrochemical chiral discrimination of the Tyr enantiomers. Other chiral amino acids including tryptophan and phenylalanine can also be successfully discriminated with the β-CD/AgNPs, suggesting high universality of the developed chiral sensor.