To valorize the excess galactose resources and prepare value-added chemicals for applications in the food and biomedical industries, Gluconobacter oxydans was employed to realize the cascaded and selective bio-preparation of galactose-derived acids. The bifunctional regulatory mechanism of Ca was the in-situ controllable preparation of calcium galactoate and 2-ketogalactonate by cleverly employing solubility difference. The presence of Ca could additionally intensify bioconversion efficiency, leading to not only increased bioconversion rate to 1.5 g/L/h, but also improved yield by 18 %. Finally, by combining enzyme hydrolysis, yeast and bacterial fermentation, 100 g cheese whey powder was effectively bio-transformed into 22.2 g bioethanol, 16.8 galactonic acid and 34.4 g 2-ketogalactonic acid, with glucose and galactose utilization rate of 83.4 % and 97.1 %. Overall, this cascading and precise regulation process not only provided a reliable idea for the downstream outlet of galactose, importantly, but also established a potential production technology for 2-ketogalactonic acid.