Abstract: The preparation and properties of cross- linked enzyme aggregates ( CLEAs) of β- galactosidase from Kluyveromyces lactis were investigated.Furthermore, the β- galactosidase CLEAs was applied to the preparation of galactooligosaccharides ( GOS) using lactose as the substrate. The optimal conditions for preparation ofβ- galactosidase CLEAs were determined as 20 times dilution of original enzyme liquid, 1 ∶ 1 of 2- propanol ( v / v) precipitation for 30 min at 4° C, and cross- linking of 30 min by 0.125% ( v / v) glutaraldehyde. Thus 45.77% of the reservation activity could be obtained.Compared with the free enzyme, the optimal p H of β- galactosidase CLEAs increased from 7 to 8, and the optimal temperature of β- galactosidase CLEAs increased from 30℃ to 37℃.Therefore, the CLEAs had wider range of p H and temperature. Meanwhile, the thermal stability of CLEAs was two times of free enzyme's. In addition, the selectivity of β- galactosidase CLEAs for GOS synthesis was higher than that of free enzyme, which was more suitable for the preparation of functional GOS.