Using N-α-benzoyl-l-arginine p-nitroanilide (BApNA) as substrate, trypsin-like enzymes (TLEs) were purified from mysis (Neomysis japonica) following two chromatographic steps, Sephacryl S100 HR gel filtration and Benzamidine-Sepharose 4B affinity. They presented a high stability in the raw material, retaining over 45% of the initial activity after 30 days of storage at pH 8.0, 45 °C. The purified TLEs had relative molecular mass between 32 kDa and 33 kDa. With higher stability and greater activity, they had similar stability and activity profiles (pH 6.0–11.0, 15–65 °C) as bovine trypsin but had a different optimum temperature (35 °C for trypsin and 45 °C for TLEs). Similar to bovine trypsin, the purified TLEs could be activated by Ca2+ and Mg2+. And the purified TLEs also showed similar inhibitory profiles as bovine trypsin with the exception of chicken egg ovomucoid (CEOM), an effective inhibitor of bovine trypsin but less so for purified TLEs. Having TLEs with physiological efficiency 3.6 times that of bovine trypsin, the use of mysis as a source for commercial production of TLEs is discussed.