Abstract: The purpose of this paper was to acquire free sperms with high quantity from spermatophores of Macrobrachium nipponensis by tryptic digestion. ?Viable sperms of spermatophore per gram were identified by normal morphology, intact structure and not being colored by trypan blue, the optimum enzyme processing conditions were determined according to the results of sperm morphology observing and trypan blue dyeing. The research was conducted under the condition of pH 7.4 and different reaction temperatures (30℃, 35℃, 40℃ and 45℃, respectively), trypsinase concentrations (0.2%, 0.4%, 0.6% and 0.8%, respectively) and reaction times (5min, 10 min, 15 min and 20 min, respectively). The best reaction condition of trypsinase digestion method was studied by orthogonal tests and the sperm amounts which got from each gram of spermatophores were used as a criterion. According to the results of orthogonal tests, we found that trypsinase concentration was the primary factor, followed by reaction temperature and enzymatic reaction time. The best effect of trypsinase digestion was at trypsinase concentration 0.8%, time 10 min and temperature 40℃. This method of acquiring free sperms is useful for further study on sperms of Macrobrachiu nipponensis.