【Background】Emamectin benzoate is a novel antibiotic insecticide and acaricide. Its extensive use may lead to serious ecological risks. It is necessary to evaluate its biological toxicity in relation to untargeted species. 【Method】The acute toxicity of 5% emamectin benzoate on six representative untargeted species, i.e. honeybee (Apis mellifera), Japanese quail (Coturnix japonica), zebra fish (Danio rerio), silkworm (Bombyx mori), Daphnia magna and earthworm (Eisenia fetida), was tested in laboratory. 【Result】The results showed that the LC50 (48 h) for acute oral toxicity (microemulsion) on bee was 0.555 a.i.mg·L-1 (highly toxic). The LD50(7 d) for quail was 148.369 a.i.mg·kg-1 (moderately toxic); the LC50 (96 h) for zebra fish was 0.368 a.i.mg·L-1; the LC50 (96 h) for silkworm was 0.005 a.i.mg·L-1 (extremely toxic), the EC50 (48 h) forDaphnia magna was 0.020 a.i.mg·L-1 (extremely toxic); and the LC50 (14 d) for earthworm was 18.397 a.i.mg·kg-1 (weakly toxic). 【Conclusion and significance】In general, the emamectin benzoate can be considered of high risk to untargeted organisms and should be used with caution.