Saccharomyces cerevisiae cannot assimilate xylose, second to glucose derived from lignocellulosic biomass. Here, the engineered S. cerevisiae strains INV Sc -XI and INV Sc -XI/XT were constructed using xylA and Xltr1p to co-utilize xylose and glucose, achieving economic viability and sustainable production of fuels. The xylose utilization rate of INV Sc -XI/XT was 2.3-fold higher than that of INV Sc -XI, indicating that overexpressing Xltr1p could further enhance xylose utilization. In mixed sugar media, a small amount of glucose enhanced the consumption of xylose by INV Sc -XI/XT. Transcriptome analysis showed that glucose increased the upregulation of acetate of coenzyme A synthetase ( ACS ), alcohol dehydrogenase ( ADH ), and transketolase (TKL) gene expression in INV Sc -XI/XT, further promoting xylose utilization and ethanol yield. The highest ethanol titer of 2.91 g/L with a yield of 0.29 g/g at 96 h by INV Sc -XI/XT was 56.9% and 63.0% of the theoretical ethanol yield from glucose and xylose, respectively. These results showed overexpression of xylA and Xltr1p is a promising strategy for improving xylose and glucose conversion to ethanol. Although the ability of strain INV Sc -XI/XT to produce ethanol was not very satisfactory, glucose was discovered to influence xylose utilization in strain INV Sc -XI/XT. Altering the glucose concentration is a promising strategy to improve the xylose and glucose co-utilization.