Abstract: The purpose of this paper was to compare two methods of acquiring free sperms from spermatophores of Chinese mitten-handed crab (Eriocheir sinensis). Under the condition of pH 7.4 and different reaction temperatures (27 ℃,32 ℃,37 ℃,42 ℃and 47 ℃, respectively), trypsinase concentrations (0.05 %, 0.10 %, 0.15 %, 0.20 % and 0.25 %, respectively) and reaction times(5 min，10 min，15 min，20 min and 25 min, respectively), the best reaction condition of trypsinase digestion method (TDM) was studied by orthogonal tests and the sperm amounts which got from each gram of spermatophores was used as a criterion. According to the results of orthogonal tests, we found that enzymatic reaction time was the primary factor, and the reaction temperature and the trypsinase concentration also functioned. The maximum effect of trypsinase digesting works at temperature 37 ℃, trypsinase concentration 0.20 % and time 5 min. By using TDM the amounts of free sperms acquired from each gram of spermatophore were 7.41×108 which was significantly higher than that using HM which was only 3.33×108 and the sperm mortality rate was 4.75 % by using TDM which was significantly lower than that using HM which was 27.00 %. Furthermore, the acrosome reaction ability was detected by artificially-induced methodology and the reaction rates were 62.3 % (TDM) and 64.5 % (HM), and there was no significant difference between them. These results suggest that TDM is more effective than HM in acquiring free sperms.