Protease in silkworm gut takes an important role in the protein digesting process and development of silkworm. In order to prove the factors influencing the protease activity in silkworm gut, we isolated the protease enzyme from the contents of silkworm gut, and determined the changing process of protease activity under different temperatures, pH values and metal ion concentrations by Folin phenol method, so as to make sure the most suitable conditions for the protease in silkworm gut. At the same time, the change of protease activity in midgut of silkworm with the increase of age was researched. The results indicated that the maximum activity of protease were at 35℃, and pH 10.1. The low concentrations of Mn²⁺, Fe²⁺ and Cu²⁺ and the high concentration of Ca²⁺ inhibited enzyme activity, while the concentration of Mg²⁺ showed significant effect on the enzyme activity. The protease activity in midgut increased greatly with the growth of silkworm.