Abstract: Penicillin hydrolysis by penicillin acylase was carried out in reversed micelle with ［C₁₄mim］Cl ionic liquids. The experiment indicated that this catalytic reaction showed better performance in enzymatic solubility, activity and stability than the reaction in reversed micelle with cetyl trimethyl ammonium bromide (CTAB). For example, the maximum solubility of penicillin acylase in ionic liquids system was twice more than that in CTAB, and the maximal enzymatic activity in ionic liquids was 1.3 times than that in CTAB.The outstanding properties of ionic liquids, including low charge density and irregular sharpness of head ring,might be attributable to its distinct head group. Ionic liquids made it possible to optimize the enzymatic catalysis in reversed micelle through altering its interfacial structure effectively.