Objective To establish a highly efficient method for the extraction, separation and purification of rat tail collagen. Methods The rat tail tendon has been obtained through stripping the rat tail; the rat tail collagen stoste has been developed through processing with Tris-HCl buffer, pepsin; sodium chloride solution has been repeatedly used for graded salting-out; acetic acid has been used for the purification of collagen. To get purified rat tail collagen, inorganic salt has to be removed with ultra-pure water dialysis. Appraisal is to be made through such technical means such as SDS-PAGE protein electrophoresis, amino acid content analysis. Results Rat tail collagen of high purity can be developed with the method established in this study, whose purity can reach electrophoresis level. When compared with those commercialized rat tail collagen products imported from overseas there is no difference. The effects the parameters for the extraction, separation and purification have exerted on yield and purity has been deliberated, established The optimal conditions for the extraction of rat tail collagen has been established, pepsin usage: 1:500, enzyme solution time: 72h, salting-out concentration: 2mol/L, the acid solution used for extraction: 0.05 mol/L acetum. Conclusion For rat tail collagen enlargement production to provide the proper processing parameters have been provided for the enlargement of production of rat tail collagen, for a large gain rat tail collagen and deeper efficacy research provides the theoretical support and practical basis have also been provided for the obtaining of large quantities of rat tail collagen so as to carry out deeper research on its efficacy.