The separation and removal of stevioside from natural product steviol glycosides to obtain high-purity rebaudioside A is of great significance for the application of steviol glycosides in food, medicine, and other fields. Here, in order to explore the adsorbent pore structure suitable for the separation of stevioside and rebaudioside A, a hierarchically porous amino-functionalized metal-organic framework (HP-NH 2 -MIL-53) with an appropriate and narrow pore size distribution was prepared using a modulator-induced defect-formation strategy. The results showed that the hierarchically porous structure with micropores and mesopores increased the specific surface area and exposed amino groups compared with original metal organic framework (NH 2 -MIL-53), and the maximum adsorption capacity of HP-NH 2 -MIL-53 for stevioside and rebaudioside A was 233.89 mg/g. The narrow pore size distribution close to 3.80 nm promoted the screening effect, resulting in a maximum adsorption selectivity of 4.13. This work proves that when the pore size of the adsorbent is between 1.41 and 3.80 nm, it has a certain pore size screening effect on stevioside and rebaudioside A, and the hierarchically porous metal-organic frameworks provide a pre-design idea of adsorbent structure for the separation of natural products with molecular weight of 800-1000 Da.