Objective: To investigate the extraction process of tea polyphenols from Xinyang Maojian with ultrasound-assisted deep eutectic solvent (DES), the antioxidant activity in vitro and in vivo and its ability to regulate intestinal flora. Methods: The single factor orthogonal method was used to optimize the extraction process of polyphenols from Xinyang Maojian tea with ultrasound-assisted DES. The scavenging ability of 1, 1-diphenyl-2-trinitrophenylhydrazine (DPPH), hydroxyl radicals and 2, 2-diazine-bis (3-ethyl-benzothiazole-6-sulfonic acid) diamiammonium salts (ABTS) was used as the antioxidant method in vitro. At the same time, the antioxidant activity of tea polyphenols at cellular level was determined by cellular antioxidant activity (CAA) method. In addition, 60 healthy male C57BL/6 mice were randomly divided into 6 groups: blank group (NC), model group (MC), positive control group (PC), tea polyphenol low dose group (LTP, 5.0 mg/kg bw), tea polyphenol medium dose group (MTP, 10.0 mg/kg bw) and tea polyphenol high dose group (HTP, 20.0 mg/kg bw). Except the NC group, the other mice were intraperitoneally injected with 50 mg/kg bw D-galactose to establish the aging model. After administration, the liver and kidney organ index, serum biochemical index, serum immune index and serum oxidation index were measured. Moreover, 16S rRNA gene high-throughput sequencing was used to cluster and analyze the diversity of cecum contents in each group. Results: The optimal extraction process of polyphenols from Xinyang Maojian tea was as follows: DES choline chloride: acetic acid 1:3, moisture content 30%, ultrasonic temperature 40℃, ultrasonic time 25 min, ultrasonic power 115 W, solid-liquid ratio 1:35 (g:mL). In addition, studies on antioxidant activity in vitro showed that when the concentration of tea polyphenols was 6g/L, the scavenging rates of DPPH free radicals, hydroxyl free radicals and ABTS free radicals were 59.23%, 60.49% and 41.65%, respectively. CAA analysis showed that when the concentration of tea polyphenols was 5g/L, CCA value could reach 63.2. The results of animal experiments showed that Xinyang Maojian tea polyphenols can significantly reduce the levels of low density lipoprotein cholesterol (LDL-C), aspartate aminotransferase (AST) and glutamic-pyruvic aminotransferase (ALT) in serum, and increase the level of high density lipoprotein cholesterol (HDL-C), which has a certain protective effect on the organs of aging mice. Moreover, tea polyphenols can increase the levels of pro-inflammatory factors interleukin-12 (IL-12), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), and reduce the levels of anti-inflammatory factor interleukin-10 (IL-10), so as to improve the inflammatory response of aging mice. Tea polyphenols also increased the activity of glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) in serum, while decreased the level of malondialdehyde (MDA), thereby increasing the antioxidant capacity of mice. Intestinal flora analysis showed that tea polyphenols can change the abundance and diversity of intestinal flora, especially increase the abundance of beneficial bacteria Lactobacillus, Candidatus_Saccharimonas and Corynebacterium, and decreased the abundance of harmful bacteria Desulfovibrio and Muribaculaceae, thereby improving the disorder of intestinal flora in D-galactose-induced aging mice, and thus effectively regulating the normal operation of metabolic pathways in mice. Conclusion: This study provides a green and efficient method for extracting polyphenols from Xinyang Maojian tea, and the polyphenols have certain antioxidant activity and the ability to regulate intestinal flora, aiming to lay a foundation for further research and industrialization of Xinyang Maojian tea.