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Trypsin from the pancreas of Tibetan sheep was purified to 7.4-fold by ammonium sulphate and acetone precipitation, followed by Sephacryl S-200 (Whatman, Maidstone, England) and Sephadex G-75 (Whatman) gel filtration, with an 23.2-fold increase in specific activity and 13.6% yield. The final enzyme preparation was nearly homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and the molecular weight of the enzyme was estimated to be approximately 27 kDa by SDS-PAGE. Trypsin-like enzymes had maximal activities at around pH 9.0 and 60C for the hydrolysis of Nα-ρ-Tosyl–L–arginine methyl ester hydrochloride (TAME). Trypsin was unstable above 60C and below pH 4.0, and was stabilized by calcium ions. Purified trypsin had a Michaelis–Menten constant (Km) and catalytic constant (Kcat) of 0.53 mM and 206 s−1, respectively, when TAME was used as the substrate. The specific trypsin inhibitors, soybean trypsin inhibited, Nα-ρ-tosyl-L-lysine chloromethyl ketone and phenyl methyl sulfonyl fluoride, strongly inhibited the activity of trypsin, while other protease inhibitors exhibited negligible inhibition. The result suggests that major proteinase in the pancreas of Tibetan sheep was trypsin-like serine proteinase. PRACTICAL APPLICATIONS Tibetan sheep is one of the unique livestock resources in western China. It was found that the trypsin extraction and purification from waste pancreas have the biological characteristics of the heat resistance, alkali resistance, high specific activity and high hydrolysis efficiency. It does not only serve as a better research tool enzyme, but is also widely applied in the food, pharmaceutical, textile and other industries. This paper provides the scientific research basis of the development of the waste pancreas for the Tibetan sheep, therefore reducing environmental pollution.