Abstract: To optimize the extraction process of polysaccharides from Diaphragma juglandis Fructus and clarify its antibacterial activity, Diaphragma juglandis Fructus was used as the raw material. Polysaccharides were extracted using the ultrasonic-assisted hot water extraction method. The effects of solid-to-liquid ratio, ultrasonic time, ultrasonic temperature, and ultrasonic power on the polysaccharide extraction yield were investigated through single-factor and orthogonal experiments to optimize the extraction process. The polysaccharide content was determined by the phenol-sulfuric acid method, and the monosaccharide composition was analyzed by high performance liquid chromatography. Using Escherichia coli and Staphylococcus aureus as test bacteria, the antibacterial activity of the polysaccharides was investigated by the growth curve method and inhibition zone assay. The results showed that the optimal extraction conditions for polysaccharides from Diaphragma juglandis Fructus were as follows: solid-to-liquid ratio of 1∶15(g/mL), ultrasonic temperature of 60 ℃, ultrasonic power of 350 W, and ultrasonic time of 40 min. Under these conditions, the crude polysaccharide extraction yield reached 15.63%, and the polysaccharide content was 80.5%. HPLC analysis revealed that the polysaccharides were composed of mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose and arabinose. The polysaccharides exhibited inhibitory effects on both E. coli and S. aureus, with a more pronounced inhibitory effect on S. aureus. This study provides a reference for the development and utilization of polysaccharides from Diaphragma juglandis Fructus and their potential application in the field of food preservation.