The enrichment and separation of astragalosides I-IV (AGs I-IV) were studied on eight macroporous resins in the present study. SA-3 resin offered the best adsorption and desorption capacities for AGs I-IV than other resins. The models of adsorption kinetics were investigated in order to elucidate the mechanism of adsorption. The pseudo-second-order model was the better choice than the pseudo-first-order model to describe the adsorption behavior of AGs I-IV onto SA-3 resin. The equilibrium experimental data were well fitted to Langmuir and Freundlich isotherms. SA-3 resin adsorption chromatography tests were carried out to optimize the separation process of AGs I-IV from Radix Astragali extracts. With the optimum parameters for adsorption and desorption, the contents of AGs I-IV were 8.78-, 11.60-, 10.52- and 11.28-fold increased with the recovery yields being 65.88, 90.92, 84.25 and 94.17%, respectively. The preparative enrichment and separation of AGs I-IV from Radix Astragali extracts can be easily and effectively achieved by SA-3 resin adsorption chromatography. The developed methodology can also be referenced for the separation of other active constituents from herbal materials and manufacture of Radix Astragali products.