Abstract：Six ionic liquids consisting different 1,3-dibutylimidazolium cation and hexafluorophosphate () or bis(trifluoromethanesulphonyl)imide (TFSI－) anion were designed and synthesized in the laboratory. The resolution of (R,S)-1-phenylethanol by Pseudomonas cepacial lipase was chosen as the model reaction to study on effects of the reaction medium, water content and temperature on the reaction. The results showed the activity of lipase in the ionic liquid 1,3-di-isobutylimidazolium hexafluorophosphate abbreviated as [D(i-C4)Im][PF6] has obviously better activity and reactivity than these in other ionic liquids and hexane. Accordingly, the ionic liquid [D(i-C4)Im][PF6] was chosen as the medium for the reaction. Under optimal condition, initial rate of lipase, the conversion of 1-phenylethanol, ee value of (R)-1-phenylethyl acetate and half life time of the lipase was 1.93 μmol•mg－1•min－1, 50%, 99% and 348 h, respectively. Moreover, circular dichroism, fluorescence spectrum and optical microscope analysis have demonstrated after the incubation of lipase in the ionic liquid [D(i-C4)Im][PF6] for 6 d the lipase exhibited an slightly increasing exposure level of amino-acid residue, however, its secondary structure is of excellent stability also and exists as its native ball enzyme conformation.