The aim of this paper was to obtain pure mono-hydroperoxide of trilinolein, which was a substance we required for the investigation of the mechanism of meat flavor formation via the technology of controlled fat oxidation combined with the thermal reaction. The material containing mono-hydroperoxide of trilinolein was prepared by oxidization of sunflower oil at 70 oC for 70 h with air ventilated at 0.72m3/kg?h, whereby the percentage of mono-hydroperoxide of trilinolein in the resulting oxidized sunflower oil was 1.95% identified by HPLC-MS and quantified by HPLC-ELSD. High-speed counter-current chromatography (HSCCC) was utilized for separation and isolation of mono-hydroperoxide of trilinolein from the oxidized sunflower oil. The optimum two-phase solvent system selected was n-hexane-dichloromethane-acetonitrile (4:1:3, V/V/V). With the upper phase of the two-phase solvent system as the stationary phase, and the lower phase as the mobile phase, the mobile phase was eluted in the normal direction at a flow rate of 1.5 mL/min while the revolution speed was set at 850 rpm/min. By the above conditions, 53 mg of mono-hydroperoxide of trilinolein was obtained in 195 min in one run from 5 g of the oxidized sunflower oil in a purity of 96.3% analyzed by HPLC-ELSD, and the recovery was 52.3%.