The viability assessment of spermatozoa in the swimming crab Portunus trituberculatus was carried out by means of the traditional biostaining techniques of trypan blue and eosin B and the method of artificial induction of acrosome reaction. With the trypan blue staining，we could not differentiate clearly between the dead and live spermatozoa. But we could distinguish clearly the difference of the dead and live spermatozoa with the eosin B staining. The morphological and structural characters of spermatozoa dyed by eosin B were studied in detail with light microscopy. The sperm has a top-like shape, and consists of an acrosome, nuclear cup and 5-10 radial arms which extend from the nucleus. The live spermatozoa were colorless, while the acrosome and nuclear cup of dead spermatozoa were stained easily by eosin B, and the boundaries of dead spermatozoa were not clear. The best concentration of eosin B and staining time were 2% and 2 min respectively. The acrosome reaction of spermatozoa were induced artificially by treatment with divalent cation ionophore A23187. And (92.73 ± 2.43)% of the emendatory rate of acrosome reaction was achieved when sperms isolated from the seminal receptacles of the female crab were exposed to 30 μg·mL^-1 ionophore A23187 for 50 min. There were significant positive correlation among the pratical value of eosin B staining、the pratical value of artificial induction of acrosome reaction and the theoretical value (P<0.01). It is concluded that both the method of eosin B staining and the method of artificial induction of acrosome reaction by ionophore A23187 were suitable for the viability assessment of spermatozoa in P. trituberculatus, and the results of these two methods were comparable.