Abstract: The triple helix structure is the key feature of collagen, and the denaturation of collagen will destroy the triple helix structure and seriously affect its biological functions. Yak is a semi-wild cattle species unique to alpine regions, and it mainly lives in the Qinghai-Tibet Plateau. We developed a mild biological process to extract high-quality yak collagen from yak bones. Polyacrylamide gel electrophoresis (SDS-PAGE) and circular dichroism (CD) results showed that the extracted type I yak collagen was not degraded, and the triple helix structure was maintained. The enzyme digestion experiment further showed that the extracted Yak ossein was not denatured, and it can effectively resist the degradation by pepsin. Meanwhile, we established a targeted binding experiment of denatured collagen, using highly specific fluorescent peptide probes to detect the denaturation level of yak collagen. The targeting probe had no binding ability to the extracted Yak ossein, indicating that the Yak ossein is non-denatured collagen. Cell experiments showed that Yak ossein had good adhesion to fibroblasts and can significantly promote cell proliferation.