Grape seed polyphenols were extracted from Cabernet Sauvignon grape seeds after winemaking. The static and dynamic adsorption-desorption capacity of AB-8 macroporous adsorption resin on grape seed polyphenols was studied， and the technological conditions suitable for the separation and purification of grape seed polyphenols were selected. On this basis， human fecal fermentation model was constructed in vitro to explore the effects of different concentrations of grape seed polyphenols on intestinal flora. The results showed that the optimal process conditions for purification of grape seed polyphenols by AB-8 macroporous adsorption resin were as follows: Loading mass concentration 8 mg/mL， ethanol volume fraction 80%， loading flow rate 2 mL/min， elution flow rate 1 mL/min. Under these purification conditions， the purity of polyphenols increased from 32.16% to 80.35%. Gut microbes sequencing results showed that the grape seed polyphenols concentration changes will not significantly affect the intestinal bacteria (P>0.05)， and will significantly influence the diversity of intestinal flora(P<0.05). At phylum level， after 24 h of fermentation， compared with blank control group (CK)， the relative abundance of Proteobacteria and Actinomycetes was increased in groups with different concentrations of phenol， while the relative abundance of Bacteroidetes and Firmicutes was decreased. However， F/B values in low concentration group (LG)， medium concentration group (MG) and high concentration group (HG) were decreased by 8.21%， 39.81% and 57.49%， respectively. At the genus level， the change of polyphenol concentration can affect the growth and proliferation of different intestinal flora， which could be shown as promoting or inhibiting the flora with the increase of the concentration， such as increasing the relative abundance of Escherichia/Shigella， Klebsiella， Lactobacillus， Bifidobacterium， Streptococcus and reducing the relative abundance of Bacteroides， Clostridium XlVa， Enterococcus， Bilophila and other bacterial genera. Concentration effects were observed with changes in polyphenol mass concentration， and different fermentation time had obvious fluctuation. Therefore， different mass concentrations of grape seed polyphenols could improve the intestinal environment by regulating different bacterial community composition， and the low mass concentration group (LG， 4 mg/mL) had the best effect.