Abstract: Tilapia intestines as enzyme raw materials are studied systematically by ultrasonic extraction technology of auxiliary, electrophoresis, chromatography techniques and so on for the first time. The results showed that: after organic mashing and ultrasound-assisted extracting, 30％－70% of ammonium sulfate fractions, QFF anion chromatography and Sephedax G-100 gel chromatography, the purified protease from tilapia intestines was obtained. The purified protease specific activity is 335 U/mg and yield is 32.8%. The result of SDS-PAGE showed a single band and the subunit molecular weight of tilapia intestines protease was 28 ku. The protease optimum pH value and temperature are 8.0－8.5 and 37－42 ℃ respectively. It is stable at pH range from 7.0 to 9.0 and has good thermal stability. Enzyme K_m value and V_maxvalue are 0.605 g/L and 9.407 μg/min. Ag⁺ and Pb²⁺ can inhibit the enzymatic activity completely while Na⁺ and K⁺ without inhibition. PMSF can strongly inhibit the activity of the enzyme. Pepstatin A and urea partly inhibit the activity of the enzyme. EDTA has no effect on the enzyme while DTT can activate the enzyme activity. The protease from tilapia intestines is a kind of serine protease. Further work should be carried out on the sequences of the enzyme and its applications．