The invention provides high-purity collagen sponge with biological activity and a preparation method of the high-purity collagen sponge. High-purity collagen with the purity higher than 99% is prepared through purification procedures of special enzymolysis, self-assembly, gel chromatography, dialysis and the like of achilles tendon of animals, and the collagen sponge is prepared through a special freeze drying procedure. According to the high-purity collagen sponge with biological activity and the preparation method of the high-purity collagen sponge, the prepared collagen sponge has a complete two-time self-assembly triple-helical structure, has a flat and fine surface and soft texture, can be used for natural cavity filling and rapid hemostasis of a wound, can be used for rapidly stopping bleeding to prevent hematopedesis of the wound and has low immunogenicity, and safety of the collagen sponge is better guaranteed.