The crude extracts from skipjack intestine was analyzed for protease activity and was found to contain 4,440 U in each extract from 1 gm intestine sample. As far as the optimum condition for the protease activity was concerned, the enzyme worked best at pH 9.0, and at the temperature of 50 ^oC. Based on the equal units of enzyme activity, this tuna intestinal protease could more efficiently hydrolyze cooked tuna dark meat than two commercial enzymes tested in this study; i.e. Alcalase and Flavouzyme. Fractionation of the crude tuna enzyme through an ion-exchange column chromatography resolved into 3 major protein peaks with protease activity, arbitrarily called B1, B2 and B3. Among the three proteins, B2 gave the highest proteolytic activity and was further purified using a column of gel filtration. This major protease had a molecular size of around 26 kDa and optimally functioned at pH 9.0 and 50 ^oC. The B2 protease activity was around 90% reduced in the presence of the detergent, SDS. Nonetheless, B2 activity was only around 26% inhibited by 2 types of surfactants, namely, Triton X-100 and Tween-20, and was only slightly affected by the metal chelating agent, EDTA. This analysis has shown that the crude tuna intestinal extract can be effectively used for protein hydrolysis at relatively higher efficiency than some commercial proteases.