Abstract: The aim of this study was to develop a novel and efficient method for the extraction of polysaccharides from Morchella septimelata, to obtain highly purified Morchella septimelata polysaccharides and to assess their in vitro probiotic proliferative activities. In this study, the polysaccharides were extracted using deep eutectic solvents (DESs) to determine the optimal solvents composition and molar ratios, to optimize the polysaccharides extraction process through a one-way combination of a response surface test, and to examine the repeat extraction performance of the solvents. Additionally, the main components of the polysaccharides, in vitro proliferation of probiotics, and the acid-producing activity after purification were clarified. The results demonstrated that the optimal composition of DESs was choline chloride and citric acid (molar ratio 1:2), and that the optimal process for polysaccharide extraction was 44% water content, 62 ℃, 65 min and a 1:42 g/mL material-liquid ratio. The polysaccharide yield was 15.24%, which was 3.26 times higher than that of the same-condition water extraction. Furthermore, the solvents demonstrated superior performance in repeated extraction. After deproteinization and chromatographic purification, the polysaccharide content reached 92.4%. The purified polysaccharide has been demonstrated to serve as an effective carbon source, stimulating the growth, proliferation and acid production of Lactobacillus plantarum and Lactobacillus acidophilus. The effect of promoting the proliferation and acid production of the two probiotic bacteria was more pronounced at a mass concentration of 2%, the growth promotion of the two probiotics was stabilised after 36 hours of incubation. In comparison to water as the extraction solvent, this study presents an effective extraction solution for polysaccharide extraction from Morchella septimelata polysaccharides and probiotics product development.