Single-factor experiment and Central Composite Design (CCD) was applied to optimize the ultrasound-assisted extraction (UAE) conditions of polysaccharides from Glycine soja (CGPS), and a preliminary characterization of three polysaccharide fractions (CGPS, GPS-1, and GPS-2) and their antioxidant activities were investigated. Under the optimal conditions: ratio of liquid to solid 42.7mL/g, extraction power 293.7W, extraction temperature 68.9°C, and extraction time 34.7min, the experimental CGPS yield was 6.04mg/g. CGPS was further purified by DEAE-cellulose and Sephadex-100 chromatography to obtain two fractions (GPS-1 and GPS-2), and their monosaccharides compositions were characterized by HPLC. Fourier-transform infrared spectra (FT-IR) indicated the chemical structures of them. Moreover, they exhibited high antioxidant activities in a concentration-dependent manner in vitro. In summary, the present study suggested that UAE was a very effective method to extract polysaccharides from Glycine soja and the polysaccharides could be explored as potential antioxidant agents for medicine and function food.