The object of the research was to extract, purify and identify the type II collagen of Agkistrodon acutus. Type II collagen of A. acutus was extracted by enzyme decomposition method, and purified by ion exchange column chromatography. It was characterized by SDS-PAGE gel electrophoresis, ultraviolet spectrophotometry, infrared absorption spectroscopy and mass spectroscopy. The results showed that the size of C II was about 130 kDa. It absorbed at 223 nm. IR spectrum obtained showed that the triple helical domains of amino-acid sequences were characterized by the repetition of triplets Gly-X-Y. The MS spectrum graphically stated that C II extracted from cow and A. acutus have the similar peptides. The C II of A. acutus was obtained by extraction and purification. Appraisal analysis by SDS-PAGE, UV, IR and MS, C II of A. acutus was consistent with the standard C II of cow. It was proved that the extracted protein was C II.