The invention discloses collagen with a triple-helix structure and a preparation method thereof. The preparation method comprises the following steps: dissolving collagen prepared by using a fermentation method in purified water, adding a mixed solution of cane sugar, dimethyl sulfoxide, glycerol, monopotassium phosphate and calcium chloride, carrying out uniform mixing, rapidly cooling the mixed solution from normal temperature to -20 DEG C by adopting a temperature program within 5 min, maintaining the mixed solution at -20 DEG C for 1 hour, then slowly heating the mixed solution to 10 DEG C by adopting a linear temperature increasing program in a period of 2 hours, keeping the mixed solution at 10 DEG C for 1 hour so as to obtain a solution of the collagen with the triple-helix structure, removing cane sugar, monopotassium phosphate and calcium chloride via washing with purified water, and carrying out spray drying or freeze drying to prepare the collagen with the triple helix structure. According to the method, the problems of animal-derived virus, nucleic acid pollution, heavy metal pollution, microbial pollution and the like in the prior art are overcome. The collagen with the triple helix structure can be used as a pure and safe biological medicine material.