Abstract: Abstract ：The cryopreservation of fishes embryos has not reached an applicational level.The main reason is the characteristics of fishes embyros, such as big physical volume, the bad permeability of vitelline membrane, etc, which casued that traditional techniques for the incorporation of cryoprotectants could not make the cyroprotectants come into the inner part of embryos sufficiently and cryoprotectants could not protect embryos effectively. But the method microinjection which was used to incorporate cryoprotectants into the yolk sac of fishes embryos, could offset the shortage of traditional methods to some extent and raise the protection degree of cryoprotectants to embryos. Several factors relating to cryopreservation of flounder Paralichthys olivaceus embryos by vitrification were studied: cryoprotectants, microinjection volumes of cryoprotectants, microinjection concentration of cryoprotectants,and the cooling sensitivity of embryos after being microinjected were analyzed. From the results, it indicated that, 1, the becoming microinjection volume was 750pl. 2, the toxicity of five single-agent cryoprotectants sequenced as follows PVP>Sucrose，DMSO>MeOH>PG, among these cryoprotectants, the embyros injected with PG got the highest survival rate and hatching rate, with PVP got the lowest, however, there was a cryoprotectant mixture PM, its toxicity was much lower than PG and the embyros injected with it got higher survival rate and hatching rate than with PG. The toxicity of cryoprotectans increased as the concentration of its increased.3, the survival rate of embryos microinjected with 6M PM was 25.07±1.57% after being coolled in -20℃ for 10 minutes by programmed cooling method, whenas ,the survival rate of controls dealt with five steps balance method was 20.88±2.84%, which indicated that the chilling sensitivity of embryos with microinjection decreased.