The effects of separation and purification on structural characteristics of polysaccharide from quinoa (Chenopodium quinoa Willd) was investigated. Using quinoa as raw material, the quinoa polysaccharide (QP) was extracted by ultrasonic-assisted method and isolated and purified to obtain three sub-fractions (QPI, QPI-I, QPI-I-I) by resin adsorption, anion-exchange and gel filtration chromatography, respectively. After separation and purification, the polysaccharide content of quinoa polysaccharide increased from 83.29% to 90.12%. XRD and Congo red experiments showed that this process contributed to the formation of crystal structure and triple helix structure. Moreover, the intensity of fourier transform infrared spectroscopy (FT-IR), 1H and 13C Nuclear magnetic resonance spectroscopy (NMR), particle size, dispersion index gradually decreased according to separation and purification, which indicated that a more uniform and stable polysaccharide structure was formed. In addition, FT-IR, 1H NMR and 13C NMR analysis showed that QP, QPI, QPI-I, QPI-I-I were all neutral polysaccharides. The above results suggested that the separation and purification process significantly enhanced the stability of polysaccharide structure. In this process, the aggregation state changed with the polarity change, and the final structure changed, which led to the change of its solubility, viscosity and other physical and chemical properties.