Corn fiber is a by-product of the corn wet milling process and a promising raw material to produce bioethanol in a bio-refinery process. In this study, enzymatic and acidic fractionations of corn fiber were compared with particular attention to produce glucose-rich hydrolyzates. The acidic fractionation contained two, sequential, sulphuric acid-catalyzed, hydrolysis steps based on our previous study. In the enzymatic fractionation process, corn fiber was pre-treated by soaking in aqueous ammonia (18.5 % (w/w) dry matter, 15 % (w/w) ammonia solution, 24 hours) and then hydrolyzed by using Hemicellulase (NS 22002) enzyme cocktail. The cellulose part of the solid residues obtained after the acidic and enzymatic fractionation processes was enzymatically hydrolyzed by using Cellic Ctec2 and Novozymes 188 (12.5 % (w/w) dry matter, 50 °C, 72 hours). Cellulose hydrolysis after the acidic and enzymatic fractionation resulted in a supernatant containing 64 g/L and 25 g/L glucose, respectively. Therefore, ethanol fermentation experiments were performed in Separated Hydrolysis and Fermentation (SHF) and Simultaneous Saccharification and Fermentation (SSF) configurations after the acidic fractionation of corn fiber. SHF configuration was found to be more advantageous regarding the achievable ethanol yield. Although the fermentation with Candida boidinii NCAIM Y.01308 was accomplished within longer time (43 hours) compared to Saccharomyces cerevisiae (5 hours), the achieved ethanol yields were similar (79%) during the SHF process. It was concluded that acidic fractionation is more efficient to produce glucose-rich hydrolyzate from corn fiber compared to enzymatic fractionation, and Candida boidinii is suitable for ethanol fermentation on the glucose-rich hydrolyzate.