Background: The recalcitrance of lignocellulosic biomass is a major limitation for its conversion into biofuels by enzymatic hydrolysis. The use of a pretreatment technology is an essential step to diminish biomass recalcitrance for bioethanol production. In this study, a two-step pretreatment using hydrothermal pretreatment at various temperatures and alkali fractionation was performed on eucalyptus fiber. The detailed chemical composition, physicochemical characteristics, and morphology of the pretreated fibers in each of the fractions were evaluated to advance the performance of eucalyptus fiber in enzymatic digestibility. Results: The hydrothermal pretreatment (100 to 220 degrees C) significantly degraded hemicelluloses, resulting in an increased crystallinity of the pretreated fibers. However, as the pretreatment temperature reached 240 degrees C, partial cellulose was degraded, resulting in a reduced crystallinity of cellulose. As compared to the hydrothermal pretreatment alone, a combination of hydrothermal and alkali treatments significantly removed hemicelluloses and lignin, resulting in an improved enzymatic hydrolysis of the cellulose-rich fractions. As compared with the raw fiber, the enzymatic hydrolysis rate increased 1.1 to 8.5 times as the hydrothermal pretreatment temperature increased from 100 to 240 degrees C. Interestingly, after a combination of hydrothermal pretreatment and alkali fractionation, the enzymatic hydrolysis rate increased 3.7 to 9.2 times. Taking into consideration the consumption of energy and the production of xylo oligosaccharides and lignin, an optimum pretreatment condition was found to be hydrothermal pretreatment at 180 degrees C for 30 min and alkali fractionation with 2\% NaOH at 90 degrees C for 2.5 h, in which 66.3\% cellulose was converted into glucose by enzymatic hydrolysis. Conclusions: The combination of hydrothermal pretreatment and alkali fractionation was a promising method to remove hemicelluloses and lignin as well as overcome the biomass recalcitrance for enzymatic hydrolysis from eucalyptus fiber. In addition, the various techniques applied in this work constituted an efficient approach to understand the underlying chemical and morphological changes of the cellulose-rich fractions.