This work concentrates on a chiral separation technology named biphasic recognition applied to resolution of alpha-cyclohexylmandelic acid enantiomers by high-speed counter-current chromatography (HSCCC). The biphasic chiral recognition HSCCC was performed by adding lipophilic (-)-2-ethylhexyl tartrate in the organic stationary phase and hydrophilic hydroxypropyl-beta-cyclodextrin in the aqueous mobile phase, which preferentially recognized the (-)-enantiomer and (+)-enantiomer, respectively. The two-phase solvent system composed of n-hexane-methyl tert-butyl ether-water (9:1:10, v/v/v) with the above chiral selectors was selected according to the partition coefficient and separation factor of the target enantiomers. Important parameters involved in the chiral separation were investigated, namely the types of the chiral selectors (CS); the concentration of each chiral selector; pH of the mobile phase and the separation temperature. The mechanism involved in this biphasic recognition chiral separation by HSCCC was discussed. Langmuirian isotherm was employed to estimate the loading limits for a given value of chiral selectors. Under optimum separation conditions, 3.5-22.0 mg of alpha-cyclohexylmandelic acid racemate were separated using the analytical apparatus and 440 mg of racemate was separated using the preparative one. The purities of both of the fractions including (+)-enantiomer and (-)-enantiomer from the preparative CCC separation were over 99.5% determined by HPLC and enantiomeric excess reached 100% for the (+/-)-enantiomers. Recovery for the target compounds from the CCC fractions reached 85-88% yielding 186 mg of (+)-enantiomer and 190 mg of (-)-enantiomer. The overall experimental results show that the HSCCC separation of enantiomer based on biphasic recognition, in which only if the CSs involved will show affinity for opposite enantiomers of the analyte, is much more efficient than the traditional monophasic recognition chiral separation, since it utilizes the cooperation of both of lipophilic and hydrophilic chiral selectors.