【Background】2,4-D Dimethylamine Salt is a selective and systemic herbicide, used to control the Chenopodiaceae and Amaranthaceae weeds in the orchards, pastures, water ways, etc. This paper confirmed the toxicity of 2,4-D Dimethylamine Salt on three freshwater fish species when used to control aquatic weeds. 【Method】Acute toxicity of 2,4-D Dimethylamine Salt on Ctenopharyngodon idellus, Hypophthalmictuthys molitrixand Carassius auratus were measured using semi-static laboratory procedures to calculate the safe exposure concentrations for these species. 【Result】The results indicated that the median lethal LC50 concentrations declined with increased exposure duration after 96 h of exposure, the median lethal concentrations (LC50) of 2,4-D Dimethylamine Salt in C.idellus, H.molitrix and C.auratus were 369.27 mg·L-1, 339.20 mg·L-1 and 438.47 mg·L-1. The safe exposure concentrations of 2,4-D Dimethylamine Salt in C.idellus, H.molitrix and C.auratus were 36.93 mg·L-1, 33.92 mg·L-1 and 43.85 mg·L-1, respectively. 【Conclusion and significance】2,4-D Dimethylamine Salt can be considered as a low toxic substance for C.idellus, H.molitrix and C.auratus. Consdering the low level of toxicity of 2,4-D on C.idellus, H.molitrix and C.auratus, this study provides scientific support for the potential spraying of 2,4-D to control aquatic weeds.