To study the physicochemical properties and antioxidant activity of ethanol-graded polysaccharides of Glehnia Radix. Four kinds of polysaccharides of Glehnia Radix (GLP-30, GLP-50, GLP-70, GLP-residual) were fractionated and purified by water extraction and alcohol precipitation. Fourier transform infrared spectroscopy, high performance liquid chromatography, and gel permeation chromatography, scanning electron microscopy, and differential scanning calorimetry-thermogravimetric analysis were used to evaluate the physicochemical properties and structure characteristics of polysaccharides. The antioxidant activities were also studied. The four polysaccharides of Glehnia Radix have the typical groups of polysaccharides, but the results of differential scanning calorimetry are quite different, which may be due to the differences in the sugar chain spatial structure. The molecular weight of each polysaccharide were decreased with increasing ethanol concentration. The yield and monosaccharide content were different among different polysaccharides. The yield (5.43%) and the content of galacturonic acid (2.03%) of GLP-50 were the highest. It also has the highest oil-retaining property, and the highest antioxidant activities in DPPH and hydroxyl free radical scavenging. The ethanol fractionation method can be used to fractionate and purify the polysaccharides of Glehnia Radix. The final concentration 50% ethanol was more suitable for the preparation of Glehnia Radix polysaccharides for its stronger antioxidant activity.