Reduce the number of pathogenic microorganisms carried by living feed can effectively reduce the occurrence of aquatic fry diseases. In this study, We evaluated the impact of six disinfectants on S-type rotifer, Brachionus rotundiformis, and the cyclopoid copepod, Apocyclops royi. Consider the 24-hours survival rate and the one-week population growth rate, the safe concentrations of povidone iodine, garlic, gallnut, amoxicillin and oxytetracycline are 10, 100, 50, 250, and 500 ppm for living feed disinfection. Chlorine dioxide is a bad disinfectant, because B. rotundiformis and A. royi are extremely sensitive to chlorine dioxide. After assessing the inhibitory effect of three common pathogens (Vibrio alginolyticus, Vibrio parahaemolyticus, and Streptococcus iniae) of fourfinger threadfin, Eleutheronema tetradactylum, 100 ppm oxytetracycline is the best choice for living feed disinfection. We carried out the disinfection of the living feed reserved by Tungkang biotechnology research center, and successfully purified 9 strains of rotifers not infected with Vibrio spp. and Streptococcus spp.. We also promte them to the aquaculture industry.