To meet the growing global demand for gluconic acid as a cement and concrete retarder, inexpensive and abundant lignocellulosic materials are regarded as the most suitable alternatives to starchy materials. However, their enzymatic hydrolysate contains not only glucose but also xylose, which negatively affects the performance of gluconic acid as a retarder. Notably, glucose is preferentially bio-oxidized into gluconic acid by Gluconobacter oxydans, but gluconic acid cannot be metabolized by Candida tropicalis. Given this, an artificially designed biological cascade process, respectively employing Gluconobacter oxydans and Candida tropicalis, was established to successfully carry out glucose conversion into gluconic acid, and xylose into a single-cell protein, using the enzymatic hydrolysate of corncobs as a feedstock. This sequential fermentation process produced 95.8 g/L gluconic acid and 9.0 g/L single-cell protein from one liter of the enzymatic hydrolysate that initially contained 98.1 g/L of glucose and 25.4 g/L of xylose. The mass-balance calculation showed that approximately 280 grams of gluconic acid and 27 grams of the single-cell protein could be harvested from 1000 grams of the corncob feedstock. The results suggest that the above-mentioned two-step bioconversion method is efficient in utilizing glucose and xylose from lignocellulosic hydrolysates.