A method for the isolation of senkyunolide I from Chuanxiong with reversed-phase preparative high performance liquid chromatography (HPLC) is described. After 95% ethanol extract was obtained with solvent extraction and normal-phase silica column separation, a fraction containing senkyunolide I was acquired and subjected to preparative HPLC for the isolation of target component. In the experiment a solvent system consisting of methanol-0.05 mol/L NH4Ac aqueous solution was found effective to eliminate the interference of ferulic acid that was the major compound in the sample. Senkyunolide I and ferulic acid were separated completely by using a stepwise gradient solvent system of 10% methanol in 10 min, and 60% methanol in 10 - 40 min. The subfractions were collected and subjected to an open MCI gel CHP-20P column for desalination respectively. Senkyunolide I was obtained and the structure was identified by mass spectrometry (MS) and nuclear magnetic resonance (NMR). This method is effective and reliable for the preparation of senkyunolide I from Chuanxiong.