Whey from cheesemaking is an environmental contaminant with a high BOD, containing an abundance of lactose. Hence, it has the potential to be utilized in the manufacturing of bio-based chemicals that have increased worth. A designed sequential fermentation approach was employed in this research to convert enzymatic hydrolysate of cheese whey (which primarily consists of glucose and galactose) into gluconic acid and bio-ethanol. This conversion was achieved by utilizing Gluconobacter oxydans and Saccharomyces cerevisiae.Glucose in the enzyme hydrolysate will undergo preferential oxidation to gluconic acid as a result of the glucose effect from Gluconobacter oxydans. Subsequently, Saccharomyces cerevisiae will utilize the remaining galactose exclusively for ethanol fermentation, while the gluconic acid in the fermentation broth will be retained. As a result, approximately 293 g gluconic acid and 103 g ethanol could be produced from 1 kg of cheese whey powder. Simultaneously, it was feasible to collect a total of 140 g of blended protein, encompassing cheese whey protein and cellular protein.Two-step fermentation has proven to be an effective method for utilizing cheese whey in a sustainable manner and reducing the environmental impact.