Using the residue after extraction of Auricularia auricula polysaccharides, the optimal process of Auricularia auricula dietary fiber (AADF) was investigated by ultrasonic synergistic enzymatic digestion, and the hypoglycemic effect of AADF was further explored by in vivo experiments. The results showed that AADF was best prepared under the process of the material-liquid ratio of 1:15 g: mL, ultrasonic power of 350 W, ultrasonic time of 50 min, and high-temperature-resistant α-amylase dosage of 5% (as a percentage of the mass of black fungus residue), with an extraction rate of 69.18%, which was superior to that of the traditional alkaline extraction method. The results of in vivo tests in mice showed that gavage of AADF had no abnormal effects on the physiological state of mice, and the growth of body mass and the glossiness of fur were abnormal. The hypoglycemic effect in diabetic mice showed that under the intervention of AADF, compared with the model group mice, the fasting blood glucose value of the rest of the gavage dose group mice was decreased by 13.72%, oral glucose tolerance was reduced by 16.95%. The total blood lipid cholesterol and triglycerides of diabetic mice were significantly reduced, and the content of high-density lipoprotein cholesterol was increased. Glutathione peroxidase in serum, catalase in the liver, succinate dehydrogenase activity significantly increased. Histopathological analysis of liver also showed that AADF had a certain protective and repairing effect on liver injury in diabetic mice. The present experimental study demonstrated that Auricularia auricula dietary fiber could effectively improve the physiological indexes of diabetic mice and provided a reference for further research and rational development of Auricularia auricula dietary fiber.