Whey generated in cheese manufacturing poses serious environmental issues that limit process profitability. The innovation in the dairy sector recognizes the "bio-refinery" as a key to successful handling of whey disposal and economic rise. Cheese whey valorisation is a complex process involving multiple technologies that might lead to value-added products (biomass, fine or bulk chemicals). This work focuses on the optimization of a fermentation process using whey as growth medium and carbon source. Lactose, which is abundant in whey, is a valuable carbon source. However, the microorganism more widely used in industrial fermentation processes, the yeast Saccharomyces cerevisiae, is not a lactose-fermenting yeast. We set up an innovative biotechnological process for the production on large scale of a not-genetically modified yeast biomass that can be used in different contexts, such as bread making, production of probiotics, nutraceuticals, bio-active molecules. In order to use the cheese whey as raw material for the cultivation of S. cerevisiae and to overcome the limitations in the use of lactose we used and externally added the enzyme ß-galactosidase. The careful optimization of the amount of added enzyme allowed the gradual release by hydrolysis and the simultaneous consumption of glucose and galactose with a consequent decrease of ethanol and an increase of the biomass produced.