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Bioethanol is an important biofuel which can be produced from the abundant low-value lignocelluloses. However, the highly toxic inhibitory compounds formed in the hydrolysate and the ineffective utilization of xylose as a co-substrate are the primarily bottlenecks that hinder the commercialization of lignocellulosic bioethanol. In this study, aiming to properly solve the above obstacles, an engineered Saccharomyces cerevisiae strain was constructed by introducing the xylose reductase (XR)–xylitol dehydrogenase (XDH) pathway, overexpressing the non-oxidized pentose phosphate pathway, and deleting aldose reductase GRE3 and alkaline phosphatase PHO13 using a GTR-CRISPR system, followed by adaptive laboratory evolution (ALE). After screening, the isolated S. cerevisiae YL13-2 mutant was capable of robust xylose-utilizing, and exhibited high tolerance to the inhibitors in undetoxified steam-exploded corn stover hydrolysate (SECSH). An ethanol concentration of 22.96 g/L with a yield of 0.454 g/g can be obtained at the end of batch fermentation when using SECSH as substrate without nutrient supplementation. Moreover, aiming to simplify the downstream process and reduce the energy required in bioethanol production, fermentation using fed-batch hydrolyzed SECSH containing higher titer sugars with a YL13-2 strain was also investigated. As expect, a higher concentration of ethanol (51.12 g/L) was received, with an average productivity and yield of 0.71 g/L h and 0.436 g/g, respectively. The findings of this research provide an effective method for the production of bioethanol from lignocellulose, and could be used in large-scale applications in future works. [ABSTRACT FROM AUTHOR]