The components and solvent properties of ionic liquids(ILs) show an important impact on papain-catalyzed asymmetric hydrolysis of D,L-p-hydroxyphenylglycine methyl ester(D,L-HPGME). The enzyme was more active, enantioselective and stable in the CnMIm·BF4(n=2—6)-based systems, while it was less active, enantioselective and stable in the systems involving C4MIm·HSO4, C4MIm·Cl, C4MIm·NO3 or C4MIm·CH3COO. Papain's activity increased with increasing the polarity of the ILs CnMIm·BF4(n=2—6) and, conversely, decreased with increasing the polarity of the ILs C4MIm·HSO4, C4MIm·Cl, C4MIm·NO3 or C4MIm·CH3COO. Also, the more hydrophobic the IL was, the more enantioselective and stable the papain was. Fluorescence spectroscopic analysis demonstrates that ILs had an obvious effect on papain's conformation. In the CnMIm·BF4(n=2—6)-containing systems, a blue-shift of the maximal emission(λmax) of papain occurred, while a red-shift of λmax took place in the systems involving C4MIm·HSO4, C4MIm·Cl, C4MIm·NO3 or C4MIm·CH3COO. Papain's conformation changed greatly when the enzyme was placed in C4MIm·HSO4, C4MIm·Cl, C4MIm·NO3 or C4MIm·CH3COO-based systems compared to those with CnMIm·BF4(n=2—6), resulting in more exposure of hydrophobic regions of the enzyme.