High-speed counter-current chromatographic preparative separation of 2-phenylbutyric acid (2-PBA) was successfully proposed. The two-phase solvent system was composed of n-hexane/butyl acetate/aqueous phase (7:3:10, v/v/v). Factors influencing the chiral separation efficiency (e.g. types and concentrations of chiral selector (CS), pH of aqueous phase and temperature) were investigated. A mathematical model was established and verified by conducting experiments. Results showed that the model predictions were in good agreement with the experimental results. Under optimal conditions, 2-PBA was successfully separated. The monomer purity reached 99.5%, and the recovery of enantiomers was 91-93%.