With the increasing applications of ionic liquids (ILs), the toxicity of ILs has drawn increasing attention in recent years, especially the influences of different anions and alkyl-chain lengths on the acute toxicity to aquatic organisms. We performed a study on the acute toxicity of 1-alkyl-3-methylimidazolium nitrate ([C(n)mim]NO3] (n = 2, 4, 6, 8, 10, 12)), 1-hexyl-3-methylimidazolium ILs ([C(6)mim]R (R = Cl-, Br-, BF4-, PF6-)) to zebrafish (Danio rerio). We also evaluated the sensibility of the investigated animals and the stability of ILs in water via high performance liquid chromatography (HPLC, Agilent 1260, Agilent Technologies Inc., USA) to prove the reliability of the present study. The results illustrated that the test zebrafish (Danio rerio) were sensitive to the reference toxicant and that the investigated ILs in water were stable. The 50% lethal concentration (LC50) was used to represent the acute toxicity to zebrafish (Dario rerio). The present study showed that the highest toxic IL is [C(12)mim]NO3 and the lowest toxic IL is [C(2)mim]NO(3)on Danio rerio. The LC(50)s for ILs with different anions had similar values. Accordingly, we believe that ILs with different alkyl-chain lengths cause greater effects than other anions on acute toxicity to aquatic organisms. Furthermore, the present study can also provide scientific methods for future studies to select and assess ILs.