The adsorption characteristics between three isoflavones in crude soybean sample and styrene-β-cyclodextrin (S-CD) were studied by molecular mechanics calculations with AutoDock. The discriminatory ability exhibited by S-CD against glycitin, daidzin, and genistin through the differences in the interaction energies and complex geometries could potentially serve for the chromatographic separation. The chromatographic elution order of the three analytes on oligo-β-cyclodextrin substituted polystyrene-based medium (PS-CDP) was predicted depending on the binding free energy values obtained from molecular docking simulations. The experimental results of chromatographic evaluation on PS-CDP were consistent with the simulation prediction. The three isoflavones in sample can be simultaneously separated in one-step under the optimized mobile phase, which consisted of methanol/0.1mM NH4AC=65.0/35.0 (v/v) by PS-CDP column chromatography. A glycitin purity of 95.1% with a recovery of approximate 86.3% was achieved by proper peak cutting, and that of daidzin and genistin was 95.8%, 95.4% and 96.2%, 95.7%, respectively.