The alkaline-extracted crude polysaccharide from Bletilla Striata (BSP-A) was used to compare the effect of protein removal by TCA method, TCA+n-butanol method, macroporous resin AB-8 adsorption method, papain method, Sevage method, and three-phase separation method. The polysaccharide retention and protein removal yield were used as important indicators, and the polysaccharide structure and biological activity were explored to determine a more suitable method for polysaccharide purification. The results showed that Sevage, papain, and three-phase separation methods could retain more than 80% of polysaccharides with relatively mild reactions. TCA method, TCA+n-butanol method, and macroporous resin AB-8 adsorption method had the best results in deproteinization, of which the highest could reach 90%. Different deproteinization methods slightly affected the molecular weight of BSP-A, while the three-phase separation method and TCA+n-butanol method affected the composition ratio of mannose and glucose of BSP-A. These six methods affected the zeta potential and FT-IR characteristics of BSP-A, among which the papain and Sevage methods could better retain the antioxidant activity. In general, Sevage method was a polysaccharide deproteinization method with good retention of polysaccharide structure and activity but low deproteinization efficiency, and it can be one of the compelling methods to remove free protein from BSP-A.