A preparative high performance liquid chromatographic(PHPLC) method was established for the simultaneous preparation of six soybean isoflavones monomers with high purity from soybean crude extracts purified with macroporous adsorption resin.The effects of experimental conditions,including mobile phase composition,flow rate and sample quantity on resolution were investigated on a SHIM-pack PRC-ODS preparative column(20 mm×250 mm,5 μm).The optimal conditions were as follows:mobile phase:acetonitrile-water by gradient elution,injection volume:800 μL,flow rate:10 mL/min.Under the optimal conditions,the baseline separation and preparation of six isoflavones monomers were achieved within 120 min.Six compounds were identified by ultra high performance liquid chromatography-tendem mass spectrometry (UPLC-MS/MS) to be daidzin,glycitin,genistin,daidzein,glycitein and genistein with purities of 95.54%,90.14%,100%,100%,96.27% and 100%,respectively.With the benefits of simplicity,good stability and high products purity,the developed method show a good application prospect in the production of soybean isoflavones standard materials.