The process parameters of extracting Tremella fuciformis Berk. polysaccharides from deep eutectic solvent (DES) were optimized in this study, including DES composition, solid-liquid ratio, ultrasonic power, ultrasonic time, and ultrasonic temperature. The results showed that the optimal extraction technology was solid-liquid ratio 1:35 (g/mL), DES water content 15%, ultrasonic time 40 min, ultrasonic temperature 60℃, ultrasonic power 200W, and the highest extraction rate was 24.73%. Subsequently, the experiment was carried out on diabetic mice with Tremella fuciformis Berk. polysaccharide obtained by this extraction method. The results showed that Tremella fuciformis Berk. polysaccharide could significantly reduce the blood glucose, oxidative stress and inflammatory response of diabetic mice, improve the body weight, glucose tolerance and insulin sensitivity of mice, and improve the state of glucose metabolism of mice. In terms of intestinal flora analysis, this study used high-throughput sequencing technology to sequence the intestinal flora of mice, and analyzed the effect of Tremella fuciformis Berk. polysaccharide on the structure of intestinal flora. The results showed that Tremella fuciformis Berk. polysaccharide could significantly increase the number of beneficial bacteria in the intestines of mice. For example, the abundance of Ddferribacterota, Verruconicrobia, Actinobacteria, Bacteroidetes and Firmicutes, while reducing the proportion of harmful bacteria, Such as Campilobacterota and Proteobacteria and so on, which helped to improve the intestinal environment of mice and improve the level of intestinal health. This study not only provided a new method for the efficient extraction of tremella polysaccharide, but also revealed the positive effects of tremella polysaccharide on the intestinal flora of diabetic mice.