The rational design of electrochemical methods for chiral recognition is a focus of research in the detection fields of biomolecules and pharmaceuticals. In this work, a novel, rapid, and convenient electrochemical approach for recognition of tryptophan isomers (L-/D-Trp) was constructed based on the alternating layer-by-layer assembly of water-soluble cationic and anionic pillar[5] arene on a carboxylic graphene (C-Gra) modified glass carbon electrode. Differential pulse voltammetry (DPV) was employed to study the electrochemical recognition of tryptophan isomers. The results reveal that both the peak currents of L-Trp and D-Trp decreased with the increasing of the layer number of the assembled pillar[5] arene, whereas the peak current value's difference between the L-Trp and D-Trp increased with the increased layers, which demonstrated an efficient route for discriminating the L-Trp and D-Trp. The recognition mechanism was studied by H-1 NMR spectra and molecular docking. The host - guest interaction is different for D-and L-Trp due to the opposite steric configurations of the Trp isomers, resulting in significantly discernible electrochemical differences in both peak currents and consequently effective chiral recognition of Trp isomers. This study is the first example that shows the construction of electrochemical chiral separation platform based on pillar[5] arene. This method shows potential applications in many fields, including separation, purification, storage of chiral molecule, drug detection and analysis. (C) 2018 Elsevier Ltd. All rights reserved.