The aim of this work was to establish optimal conditions for the enzymatic production of lactulose by beta-galactosidase from Kluyveromyces lactis using cheese whey as feedstock. Initially, the production of this disaccharide was evaluated using free beta-galactosidase and synthetic medium (containing lactose and fructose), and the optimal conditions for the production of lactulose were as follows: lactose/fructose ratio of 15/30% (w/v); total initial concentration of lactose and fructose of 200 g/L; and operational conditions at 50 degrees C, pH 7. With the addition of 0.01% w/v Mg2+, the system reached a maximum lactulose production of 22.6 g/L in 2 h of reaction time with a productivity of 11.3 g/(L.h). Then, beta-galactosidase was immobilized on chitosan activated with glutaraldehyde to obtain highly active and stable catalysts for lactulose synthesis using cheese whey and fructose as the substrate. The highest production of lactulose (17.3 g/L) was achieved with the treated cheese whey for protein precipitation and was observed at a high operational stability. It was therefore concluded that it is possible to use lactose from cheese whey for the synthesis of lactulose, which could provide an appropriate use for this waste product, reducing environmental damage and cost.