In recent decades, recovery and identification of digestive enzymes from fish has attracted much attention and new applications for these enzymes have been defined in some industries, such as the food and detergent industries. One of these recovered enzymes is Trypsin, which in this study was extracted and purified from the intestine of Caspian kutum using a three-step extraction method with 50 mM Tris-HCl buffer (containing 10 mM CaCl2 pH 7.5), 60% ammonium sulfate precipitation and Sephadex G-75 gel chromatography. The Result was an enzyme with 52% yield and a purity of 25.78-fold. The kinetic parameters of Vmax and Km for BAPNA substrate were calculated using the Michaelis-Menten equation and were 2.9958 µmol/mg.min and 0.5350 mM respectively and Kcat was 6.2568 S-1. The kutum Trypsin’s half-life was also calculated as 58.72 min for BAPNA substrate using the conversion of the enzyme activity graph (in minutes) to natural logarithmic graph and determining its slope. The results showed that it is easy and economical to extract and purify Trypsin from intestine of Caspian kutum and it is kinetically more effective than commercial Trypsin. It is suitable for use in the food industries, fish processing industry, culture media production as well as application in biotechnology research. Published