【Background】Glyphosate is a sterilizing herbicide effective in controlling perennial weeds. In recent years, glyphosate has been increasingly used to control aquatic weeds, its safety to fish is paid attention. 【Method】Acute toxicity of glyphosate on Ctenopharyngodon idellus, Hypophthalmictuthysmolitrix and Carassius auratus was investigated by using standardized laboratory procedures to calculate the safe exposure concentrations for these species. 【Result】The results indicated that the median lethal concentrations (LC50) in these fish species gradually declined over exposure time. LC50 of glyphosate on C.idellus, H.molitrix and C.auratus were 0.2518 mg·L-1, 0.2588 mg·L-1 and 0.2599 mg·L-1, respectively after 96 h exposure. The safe exposure concentrations were 0.0252 mg·L-1, 0.0259 mg·L-1 and 0.0260 mg·L-1 for C.idellus, H.molitrix and C.auratusrespectively. 【Conclusion and significance】Glyphosate showed high toxicity to these three freshwater fish species, C.idellus, H.molitrix and C.auratus. This paper indicated the importance to improve guidelines for spraying glyphosate when used in freshwater farming.