Abstract: The article was to investigate the optimal purification process for macroporous resins of polysaccharides from Rehmannia glutinosa Libosch. With weighted comprehensive scores of the rate of polysaccharide retention, decolorization and protein removal, the macroporous resin with better purification effect was selected. Then, the optimal experiments were carried out by single experiments and the Box-Behnken response surface methodology(RSM) experiment. The results showed that the mixture of HPD-400 and LS-700B macroporous resin had the best purification effect on Rehmannia glutinosa Libosch. polysaccharides, the optimal purification process was as follows: Resin mass ratio was 1.20, sample loading concentration 6.0 mg/mL, temperature 30℃, sample loading rate 1.5 BV/h, and the upper column volume 1.5 BV. After purification, the retention rate of polysaccharide was 90.25%, the decolorization rate was 83.15%, the protein removal rate was 79.75%, and the comprehensive scores was 84.97%. The purification process obtained in this study was suitable for the purification of Rehmannia glutinosa Libosch. polysaccharides.