The lactose in milk was hydrolyzed by β-D-galactosidase from permeabilized k.fragilis cells.The factors affecting the degree of lactose hydrolysis, lactose hydrolysis kinetics and the stability of this enzyme were investigated. The results showed that the percentage of lactose hydrolysis was up to 62.66% after reaction 75 min at 4 ℃. The Km value and Vmax value for k.fragilis β-D-galactosidase was 3.18 mmol/L and 3.32 μmol/min, respectively. Glucose as a hydrolysis product exhibited feedback inhibition of k.fragilis β-D-galactosidase, 36.88% of the enzymatic activity was inhibited at 50 mg glucose/mL. After 4 batches of lactose hydrolysis in milk, enzyme activity retained more than 65% of the initial enzyme activity. β-D-galactosidase from permeabilized k.fragilis cells had good storage stability at low temperature, it preserved 95.64% of initial activity after 14 days storage at 4 ℃.