Myofibril-bound serine protease (MBSP) is a unique enzyme of fish muscle, as it tightly binds to myofibrils even in the presence of detergents, and is suggested to participate in the physiological myofibrillar metabolism of fish. The soluble form of MBSP had been isolated from the myofibrillar fraction of marine fish including white croaker Argyrosomus argentatus, wanieso lizardfish Saurida wanieso, and brushtooth lizardfish S. undosquamis and freshwater fish including common carp Cyprinus carpio and crucian carp Carassius giberio langsdorfi. MBSP was highly sensitive to specific inhibitors for serine protease and catalyzed hydrolysis of synthetic substrates for trypsin. In addition, the N-terminal amino acid sequence of brushtooth lizardfish MBSP showed high homology to fish pancreatic trypsin. These findings suggest that MBSP is clearly classified into a trypsin-type proteolytic enzyme. However, MBSP was extremely stable than trypsin under the physical conditions such as wide pH and temperature ranges and in the presence of organic solvents. Brushtooth lizardfish MBSP rapidly degraded myosin heavy chain rather than other myofibrillar proteins, and significantly maintained this myofibrillar degrading activity over 24 hours under the physiological conditions. These characteristics differ from not only fish trypsin but also other fish muscular proteases including cathepsin, calpain, multicatalytic protease and known fish muscular serine proteases, indicating that MBSP is a novel type of fish muscular protease. This article introduces unique enzymatic characteristics of MBSP in comparison with fish trypsin and other muscle proteases.