Lactose in whey permeate, a by-product of dairy processing, can be converted into valuable sugars like glucose, fructose, and galactose. This study explores the use of cross-linked enzyme aggregates (CLEAs) for this conversion, focusing on β-galactosidase and glucose isomerase. These enzymes were successfully immobilized with final concentrations of 30 mg of wet CLEAs/mL and 10 mg of wet CLEAs/mL, respectively. The CLEAs demonstrated high stability and recyclability, retaining activity after seven cycles. In a batch process, immobilized β-galactosidase hydrolyzed 40 mM lactose to completion in 15 minutes at 60°C, and immobilized glucose isomerase progressively isomerized glucose into fructose, reaching an 8 mM fructose concentration after 7 hours. This work also addresses challenges in CLEA development, explores improvement opportunities in glucose isomerase cross-linking, and proposes future CLEA application alternatives in dairy by-product valorization.