Sea cucumber is easy to undergo autolysis, causing serious economic loss in the aquaculture, fishery, and food processing. However, research on the autolysis of sea cucumber is limited. In the present study, the autoenzyme that could hydrolyze the structural protein of body wall was identified and characterized in sea cucumber Stichopus monotuberculatus. Although the main edible part of sea cucumber was the body wall, its microstructure was shown to be almost composed of collagen fibers belonging to extracellular matrix, indicating that the body wall of S. monotuberculatus belonged to dermis tissue. Starting from this perspective, crude collagen fibrils from dermis of S. monotuberculatus was isolated, and pepsin solubilized collagen was also obtained by hydrolyzing collagen telopeptides from crude collagen fibrils. Crude collagen fibrils and pepsin solubilized collagen were then acting as substrates, respectively, to identify dermis autoenzyme which had hydrolytic activity on dermis collagen. After that, a collagenase of 45 kDa was identified with the method of collagen zymograph, and the content of soluble protein was suppressed by collagenase inhibitor significantly in the autolysis of dermis. Finally, the effect of environmental conditions on collagenase activity was studied, results showed its best activity was at 40°C and pH 8, and divalent metal ion Mn²⁺ was essential for its activity. As a whole, our results showed that the dermis of sea cucumber was composed of collagen fibers, and collagenase was the main enzyme resulting in autolysis of sea cucumber.