Ultrasonic-assisted extraction (UAE) and response surface methodology (RSM) were applied to the extraction of polysaccharides from the base of Flammulina velutipes stipe. Results showed that the optimum extraction parameters were ultrasonic power 680 W, time 19.8 min, the ratio of water to raw material 28 mL/g, and the yield of polysaccharides was 16.20%. After separation by DEAE cellulose-52 and Sephadex G-100 size-exclusion chromatography, three main fractions, FVSP -1, FVSP -2, and FVSP -3, were obtained. The relative molecular weights of which were 21 kDa, 224 kDa, 357 kDa, respectively, and all the three fractions were heteropolysaccharides with a glucan as backbone chain.