Abstract: The study aimed to explore the optimal conditions for the extraction of polysaccharide from Elaeagnus angustifolia flower and its effect on probiotic proliferation in vitro. Polysaccharide was extracted from Elaeagnus angustifolia flower using hot water and ultrasonication. The effects of the material-liquid ratio, duration of ultrasonication, extraction time and extraction temperature on the polysaccharide yield were analyzed. The extraction conditions were optimized by response surface methodology, and the effects of different polysaccharide concentrations (0, 0.5%, 1.0%, 1.5%, 2.0%, and 3.0%) on the proliferation and acid production of three probiotics were compared. The results showed that the optimal extraction conditions of Elaeagnus angustifolia flower polysaccharide were as follows: Material-liquid ratio, 1:25 g/mL, duration of ultrasonication, 21 min, extraction temperature, 72℃, extraction time, 62 min. The polysaccharide yield was 12.45%±0.15%, which was close to the theoretical predicted yield (12.587%). The highest OD values of Lactobacillus acidophilus, Bifidobacterium bifidum, and Bifidobacterium adolescentis were obtained at a polysaccharide mass concentration of 2%, being 1.23±0.01, 1.06±0.02, and 1.22±0.02, respectively, and the lowest pH values (5.17±0.04, 5.95±0.04, and 5.52±0.02, respectively). The growth of the three probiotics stabilized after the incubation time reached to 40 h. It indicated that Elaeagnus angustifolia flower polysaccharide promoted the proliferation and acid production of three probiotics. These findings indicate the potential of the polysaccharide from Elaeagnus angustifolia flower as a prebiotic and provide a theoretical basis for further research and the utilization of Elaeagnus angustifolia flower resources.