A new method for isolation of gentiopicroside (GPS) from Swertia mussotii Franch with reversed phase preparative high performance liquid chromatography (Pre-HPLC) is described. After 95% ethanol extract was obtained with solvent extraction and macroporous resin separation, a fraction containing GPS was acquired and subjected to Pre-HPLC for isolation of target component. The optimum operation parameters were selected as follows: a C18 column (200 mm x 50 mm, 5 microm), methanol-0.1% acetic water (30:70, v/v) as the mobile phase at flow rate of 75 mL/min, the detection wavelength at 254 nm, and the injection volume of 500 microL at the mass concentration of 225 g/L. The purity of the product was detected by HPLC method. The result showed that the purity of product was above 99%. The effective and rapid method has been successfully applied to the preparation of GPS from Swertia mussotii Franch.