Abstract: OBJECTIVE To establish a chiral ligand-exchange method by HPLC for the separation of carnosine enantiomers. METHODS Chromatography was carried out by chiral column (Phenomenex chirex 3126) with mobile phase composed of copper sulfate as chiral mobile phase ligands. The effect of sample volume, organic-additives, flow rate of mobile phase, copper concentration, column temperature and thermodynamics character were investigated. Chiral identify principle of solute was researched preliminarily. RESULTS Carnosine enantiomers can be separated on baseline with a mixture of 2 mmol·L^-1 CuSO₄-acetonitriles(96∶4) as mobile phase at a flow rate of 1.0 mL·min^-1 and detected at 254 nm. Both enthalpy change (ΔΔHo) and entropy change (ΔΔSo) have contributed to the chiral recognition. The process of chiral separation is the enthalpy-controlled. The comoles compound of L-carnosine, chiral ligands and Cu2+ present the instability, in accordance with the typical chromatographic behaviour of conformation change. CONCLUSOIN Carnosine enantiomers can be separated on baseline by chiral ligand-exchange method. The chiral ligand of L-carnosine is unstable.