β-Galactosidase catalyzes the hydrolysis of lactose to glucose and galactose. The hydrolysis of lactose is a promising process in the food industry for the development of new products with no lactose in their composition. The elimination of lactose allows the consumption of milk for the great percentage of the world population (about 70%) which suffers of lactose intolerance. Also, cheese whey is an abundant subproduct of the dairy industry that contains 50 g/L of lactose. After hydrolysis, it may be used in the food industry for development of new products with no lactose contents. The immobilization of the enzyme allows its use in continuous process and the reuse of the enzyme, what may reduce costs in the lactose hydrolysis process. Therefore, in this work, β-galactosidase from Kluyveromyces fragilis was immobilized on different activated supports. The best derivative was obtained by immobilizing the enzyme on chitosan coagulated with KOH and activated with glutaraldehyde, both steps carried out at 50°C. The immobilization yield and recovered activity were 100%. The pH and temperature of maximum activity was similar of the soluble enzyme (pH 7 and 45°C). An enzyme load as high as 247 mg of protein per gram of support could be reached. The operational stability of the immobilized enzyme was higher than the soluble one. After four cycles of lactose hydrolysis (20 minutes each), at 40°C and pH 7; the immobilized enzyme lost only 17% of the initial activity.