β-galactosidase was immobilized on chitosan cross-linked with glutaral-dehyde.The orthogonal experiment was adopted to determine immobilized carrier and the immobilization conditions.The results showed that the suitable molecular weight and concentration of immobilized carrier (degree of deacetylation of chitosan was more than 90％)were 3 × 105 and 2％ (v/v).Chitosan microsphere carrier had good ballabililty and mechanical strength.β-galactosidase was immobilized on chitosan microspheres using cross-linking method.The immobilization conditions were as follows:glutaraldehyde concentration and crosslinking time were 10 g/L and 1 h,enzyme concentration and immobilization time were 1.5 mg/mL and 12 h,the activity recovery of the immobilized enzyme reached 70.5％.At the same time,the immobilized enzyme had good storage stability and operation stability,which had certain application value.