The invention relates to a panax notoginseng seedling protection film, and a preparation method and an application thereof. The film provided by the invention is a water-soluble gel prepared by using a water-soluble film-forming material, panax notoginseng polysaccharide, a root growth promoter, a preservative, and a pesticide. With the material, a protection film can be formed on the surface of a panax notoginseng seedling. With the panax notoginseng seedling protection film provided by the invention, water loss of panax notoginseng seedling during a transplanting process can be reduced, and seedling surface skin damage caused by abrasion during transportation or during transplanting can be reduced. Therefore, fungal infection or insect attack caused by damages after transplanting can be reduced, occurrence rate of panax notoginseng root rot, sclerotinia stem rot, sheath blight, anthracnose, and blight can be reduced, and panax notoginseng survival rate can be improved. The panax notoginseng seedling protection film provided by the invention has good physiochemical performances such as film-forming performance, water resistance, air permeability, water permeability, and the like. Therefore, healthy growth of crops can be protected.