This study aimed to optimize the ultrasound-assisted aqueous two-phase extraction (UATPE) process of Anemarrhena asphodeloides polysaccharide (AAPs), separate and purify the crude AAPs by different column chromatography techniques to obtain a uniform polysaccharide fraction (AAPs-1-SG), and characterize its structure. The effect of AAPs-1-SG on the viability of RAW264.7 cells was determined by the MTT method. The neutral red method was used to evaluate the effect of AAPs-1-SG on the phagocytic activity of RAW264.7 cells, and the effects of AAPs-1-SG on the levels of NO, IL-1β, IL-6 and TNF-α in RAW264.7 cells were investigated. The results showed that the process parameters of UATPE polysaccharide from Anemarrhena asphodeloides were obtained as follows: Ammonium sulfate mass fraction of 24%, ultrasonic power of 400 W, ethanol volume fraction of 45%, and solid-to-liquid ratio of 1:20 g/mL, and the yield of AAPs was 12.17%±0.25%. Ultraviolet spectral scanning revealed that AAPs-1-SG had no proteins or nucleic acids. FT-IR showed that AAPs-1-SG exhibited typical infrared absorption characteristics of polysaccharides and possessed α and β types glycosidic bonds. AAPs-1-SG was mainly composed of Glc, Ara, and Man, and its molecular weight was 11298 Da. Scanning electron microscopy and atomic force microscopy observed that AAPs-1-SG presented irregular sheet-like and porous structures. In addition, AAPs-1-SG could markedly increase the viability and phagocytic activity of RAW264.7 cells, and significantly improve the levels of NO, IL-1β, IL-6, and TNF-α (P<0.05), further revealing that AAPs-1-SG had better immunomodulatory activity. The findings can provide an important method for the efficient extraction of natural polysaccharides and also offer significant references for the development and utilization of AAPs.