Preparative high-speed counter-current chromatography (HSCCC) was used for the isolation and purification of solanesol from potato leaves. Experimental conditions of the extraction of solanesol from potato leaves have been optimized. An ultrafine extraction method was applied in this study. The efficiency using an ultrafine extraction was found to be improved in the investigation, the yields of solanesol by different extraction methods were 0.083% by ultrafine extraction and 0.050% by ultrasonic extraction. Using n-hexane-methanol (10:7, v/v) as the two-phase solvent system, preparative HSCCC was successfully performed with the yield of 5 mg solanesol at 98.7% of purity from 60 mg of crude extract in the one-step separation. The mobile phase was the lower phase and operated at a flow rate of 1.5 mL/min, while the apparatus rotated at 800 r/min. The solanesol was identified by the atmospheric pressure chemical ionization mass spectrometry (APCI-MS). The ionization and cleavage mechanisms of solanesol in APCI-MS and APCI-MS/MS are discussed.