Objective? Zebrafish is one of the most widely used laboratory animals in scientific research, and the embryonic period is the most important application period. The hygiene of embryos directly affects development and survival rate of zebrafish, and the potential pathogens carried by embryos also affect the quality of experimental data. In this study, we tested different disinfectants and different action times to obtain an efficient protocol for zebrafish embryo disinfection, which in turn promotes the management in fish houses. Method? In this study, we tested three reagents, including reagent grade povidone iodine, reagent grade sodium hypochlorite and chlorine dioxide, that are readily available in the domestic market and can be used for embryo disinfection. We evaluated the toxic effects at three concentrations and two action times on zebrafish embryos of two ages, in term of the rates of survive, dechorion and malformation. The effects of the three disinfectants were compared from the bacteriological point of view that the amounts of bacteria on the embryo surface after disinfection. Results? It was found that chlorine-containing disinfectants, i.e. sodium hypochlorite and chlorine dioxide, were better able to kill bacteria on the surface of embryo, while povidone-iodine was not quite effective. 30 ppm sodium hypochlorite and chlorine dioxide-treated zebrafish embryos did not differ from the control group in terms of the rates of survive, dechorion and malformation. Moreover, chlorine-containing disinfectants treated for 10 minutes were more effective in sterilizing the embryos than those treated for 5 minutes. In view of the complexity of chlorine dioxide using, this study recommends treating 6 to 24 hpf embryos with 30ppm sodium hypochlorite for 10minutes as an operational method.