The invention provides a Chinese chestnut anti-cancer active polysaccharide and a separation and purification method thereof and relates to the field of natural drugs. The preparation method comprisesthe following steps: obtaining Chinese chestnut crude polysaccharide by utilizing a solvent extraction method, transferring the Chinese chestnut crude polysaccharide solution into a dialysis bag, andcarrying out dialysis, so that Chinese chestnut polysaccharide dialysate is obtained; and separating the Chinese chestnut polysaccharide dialysate by adopting column chromatography, wherein deionizedwater is taken as an eluting agent, and flow velocity of the eluting agent is 25-35mL/h. The method adopts a way that a dialysis method is combined with a column chromatography method, small molecular substances in the Chinese chestnut crude polysaccharide are removed, and the obtained product has high Chinese chestnut polysaccharide content and obvious anti-cancer activity.