Abstract: Hydrolysis of lactose with β-galactosidase is one of the most promising biotechnological applications in the food industry because of it is use in the production of low lactose dairy products. Acid whey samples were collected from the dairy pilot plant at the University of Jordan, Faculty of Agriculture. β-Galactosidase enzyme from Kluyveromyces lactis with initial activity of 3000 LAU/ml was immobilized in a sodium alginate and used for acid whey hydrolysis in fluidized bed reactor. Optimization parameters of lactose hydrolysis were (12 and 24% v/v enzyme concentration), (10 and 20 ml/mint flow rate). The process reaction was done at a temperature of 37oC and 6.5 pH. Lactose hydrolysis of 81% and 85% were obtained during 6 h of incubation for 12 and 24% v/v enzyme concentration at flow rate of 10 ml/min.