In this experiment, Crassostrea angulata sperm was used as the research object to study the effect of salinity stress on sperm quality and fertilization rate using PI/Rh123 dual fluorescent staining and flow cytometry (FCM). The results showed that sperm moving-time, survival rate and sperm activity increased first and then decreased with the increase of salinity. The suitable salinity range of C. angulata sperm was 20–35, and the salinity range was 25–35 with higher sperm activity. It was found that salinity stress caused damage to the sperm plasma membrane first and then damaged the mitochondrial activity by PI/Rh123 dual fluorescent staining and FCM. In addition, sperm were severely damaged under low salinity conditions (10, 15) and the damage ratio of sperm plasma membrane was 67.26%±2.35%, which was significantly different from the other groups. The artificial insemination experiment showed that fertilization rate and cleavage rate decreased significantly after low salinity stress (10, 15), and deformity and split stagnation were observed in cleavage stage, which shows that sperm quality not only seriously affects the process of fertilization, but also has a certain effect on cleavage. This experiment explored the feasibility of PI/Rh123 dual fluorescent staining and FCM to detect the function of plasma membrane and mitochondrial activity in oyster sperm, but also evaluated the sperm quality comprehensively, which provided a theoretical basis for the research of oyster sperm quality.