In this work,an effective method was established for the systematic separation and purification of high-purity compounds from Fructus Psoraleae by a two-dimensional reversed-phase preparation chromatography with separate-enrichment mode.After grinding extraction,the dead adsorption and liposoluble molecules were removed from the crude extract after the silica gel column chromatography and extraction.The C₁₈ column (250 mm × 30 mm,10 μm) was selected as the first and second-dimensional separation column.The water-acetonitrile was selected as mobile phases.The C₁₈ column (80 mm × 30 mm,10 μm) was selected as enriching columns and water as the diluent solution.The conditions for the first-dimensional and second-dimensional chromatographic separation were predicted and optimized using XTool chromatography expert software and HPLC.Moreover,the sample loading amount of one-dimensional chromatography separation was 500 mg and the one-dimensional separation was performed within 200 min.As a result,18 components were obtained by the first-dimensional separation and 17 compounds were isolated in more than 98% purity by the second-dimensional,which were further identified by mass spectrometry (MS) and standards,and the recovery rate of the second-dimensional preparation was 93.92%.This method has the potential to be an efficient separation method of high-purity compounds from Fructus Psoraleae and it shows great promise for the separation of high-purity compounds from other kinds of natural products.