Utilization of sweet and acid whey is a frequently researched and discussed topic. Especially, the non-protein fraction of acid whey is seldom utilized in food applications. Hence, the aim of this study was to valorize the non-protein fraction of sweet and acid whey by transgalactosylation of lactose to prebiotic lactulose. Therefore, 7 commercial β-galactosidases were tested regarding their ability to produce lactulose in sweet and acid whey UF-permeates (pH 4.5 to 6.5). Additional performance parameters characterizing the transgalactosylation reaction were defined and evaluated to select enzyme preparations for robust processing. Three promising enzyme preparations were identified, which provide a high lactulose yield (Y ≥0.2) after 48 h at 6°C. Solely Enzeco is usable for sweet and acid whey, while Biolactase and Lactozym are only usable with sweet whey. The degree of hydrolysis exceeded 0.8 only with the bacterial enzyme preparations, which showed no affinity toward the transgalactosylation reaction. Yeast β-galactosidases provided overall higher isomeric ratios of 1-lactulose, compared with fungal β-galactosidases. Subsequently, the feed composition was optimized for the enzyme preparations using a central composite design. Concentration of lactose and fructose as well as enzymatic activity were varied on 5 levels. Using a response surface analysis the optimal feed composition for sweet (13% wt/wt fructose and 6.5% wt/wt lactose) and acid (23% wt/wt fructose and 6.0% wt/wt lactose) whey could be determined.