A xylose-utilizing recombinant diploid industrial strain NAN-127 of Saccharomyces cerevisiae, containing the genes XYL1 and XYL2 from Pichia stipitis encoding xylose reductase(XR) and xylitol dehydrogenase(XDH) respectively and the gene XKS1 encoding xylulokinase(XK) for overexpression from S. cerevisiae, was constructed by integrating vector at the rDNA locus. The recombinant strain NAN-127 was stable for more than 60 generations in nonselective medium and displayed at least 9.3 times higher specific activities of these three enzymes (XR, XDH and XK) compared to the parent strain NAN-27. The results of the cofermentation with 80 g glucose L^-1 and 50 g xylose L^-1 in 5L fermentor showed that xylose consumption ratio was 65.5% by the recombinant strain NAN-127 at 72h, which was 2.8 folds compared with the parent strain, under the optimal conditions: ventilation rate 0.04 L L^-1min^-1, pH 4.5, temperature 30.0°C. Meanwhile, both ethanol and xylitol yield by the recombinant strain NAN-127 (Y _{g Ethanol / g Total sugar consumed} = 0.390 and Y _{g Xylitol / g Xylose consumed} = 0.446 ) were higher than the parent strain.