A Saccharomyces cerevisiae strain CCW12OE was constructed by overexpressing CCW12 in a previously reported strain WXY70 harboring six xylose utilization genes. CCW12OE produced an optimal ethanol yield of 98.8% theoretical value within 48 h in a simulated corn stover hydrolysate. CCW12OEwas comprehensively evaluated for ethanol production in Miscanthus, maize and corncob hydrolysates, among which a 96.1% theoretical value was achieved within 12 h in corncob hydrolysates. Under normal growth conditions, CCW12OE did not display altered cell morphology; however, in the presence of acetate, CCW12OE maintained relatively intact cell structure and increased cell wall thickness by nearly 50%, while WXY70 had abnormal cell morphology and reduced cell wall thickness by nearly 50%. Besides, CCW12OE had higher fermentation capacity than that of WXY70 in undetoxified and detoxified hydrolysates with both aerobic and anaerobic conditions, demonstrating that CCW12 overexpression alone exhibits improved stress resistance and better fermentation performance.