Abstract Three 1,3-dipentylimidazolium hexafluorophosphate ionic liquid isomers, 1,3-di-n-pentylimidazolium hexafluorophosphate ([D(n-C5)Im]PF6), 1,3-di-isopentylimidazolium hexafluorophosphate ([D(i-C5)Im]PF6), and 1,3-di(2-methylbutyl)imidazolium hexafluoro-phosphate ([D(2-mb)Im]PF6), were designed and synthesized. They were used as media for the synthesis of phenylethyl acetate catalyzed by Pseudomonas cepacial lipase. The activity and reactivity of the lipase in [D(2-mb)Im]PF6 is obviously higher than that in organic solvent hexane. Based on the temperature-controlled characteristics of [D(2-mb)Im]PF6, we designed a new route to synthesize phenylethyl acetate catalyzed by lipase, in which the reaction and the separation of the product were carried out under a relatively high and low temperature, respectively. By investigating the influence of various factors on the yield of product, optimal conditions for the synthesis of phenylethyl acetate were obtained, i.e., the amount of lipase 30 mg, the ionic liquid 1.0 g, water content 2%, reaction temperature 35 °C, and reaction time 48 h. Under the optimal conditions, the yield of phenylethyl acetate was up to 92.3%. Also, the stability of the lipase in the [D(2-mb)Im]PF6 medium was 7.4-fold that in hexane. The lipase in [D(2-mb)Im]PF6 was recycled 10 times without substantial diminution in activity. Moreover, circular dichroism and fluorescence spectroscopy were used to follow the changes of enzyme structure in different solvents. The results showed that the lipase in the ionic liquid has excellent secondary structure stability and slightly big exposure level of amino-acid residues.