The study on sulfated beta-cyclodextrin binding to uranyl ion helps to get a better understanding of uranyl compounds' intermolecular interaction mechanism and facilitates the structure-based design of uranyl binding molecules. Here we investigated the electromigration of the inclusion complex by using affinity capillary electrophoresis in acidic solution. The binding constant was determined to be logK = 2.96 +/- 0.02 (R-2 = 0.996) through nonlinear regression approach. The possible configurations and structural features of the inclusion complex were further studied by molecular dynamics simulation. The results suggest the distinctions of coordination environment and hydration compared with bare uranyl ion in aqueous solution. Thus, two water oxygen atoms coordinated with uranyl in the first hydration shell at 2.55 angstrom instead of five in the same distance range. The binding free energy was calculated as -12.10 +/- 1.46 kcal/mol by means of thermodynamic perturbation method. The negative value indicates that the process of S-beta-CD capture uranyl ion in the aqueous media is spontaneous.