Pitcher fluid of Nepenthes × Miranda is usually discarded before transportation despite containing many functional materials, in which hydrolytic proteases are particularly attractive to us. In our study, we collected the pitcher fluid of Nepenthes × Miranda and got pitcher fluid proteases with 6.658 U/mL of proteolytic activity by concentration treatment. Then the optimal pH and temperature were detected, and the activators, denaturants and inhibitors were screened respectively. Finally, Michaelis constants were calculated to quantify the affinity with different substrates. The results showed that the highest activity was at pH 2.05~2.50 and 50~55 ℃, which indicated the advantages of lower pH and higher temperature compared with the nepenthesins from other species of Nepenthes. Moreover, the pitcher fluid proteases could be activated by Mn2+ significantly and tolerated glycerol of high content. We discover a way of the new protease resources, a rational use for biochemistry and a direction for the reuse of agricultural waste.