A protocol has been developed to enhance the activity of cross-linked enzyme aggregates (CLEAs) through adding sugar during the preparation. The effect of glucose, sucrose and trehalose on the activities of CLEAs of penicillin G acylase (PGA) were investigated. The highest activity was obtained in the case of adding trehalose. Confocal laser scanning microscopy and Fourier transform infrared spectroscopy studies showed that the polar microenvironment and the secondary structure of native PGA were preserved to some extent when it was prepared as CLEAs with sugar added which result in its higher activity compared with that prepared without sugar. Scanning electron microscope revealed the different inner morphologies the kinetic studies showed the higher affinity and resist-inhibition capacity of CLEAs prepared under this protocol. Furthermore, CLEAs prepared in the present of sugar remained higher thermal stability when it was incubated at high temperature.