Co-culture of xylose-utilizing and inhibitor-tolerant Saccharomyces cerevisiae was developed for bioethanol production from undetoxified pretreated biomass in simultaneously saccharification and co-fermentation (SSCF) process. Glucose accumulation during late fermentation phase in SSCF using xylose-utilizing strain can be eliminated by the introduction of inhibitor-tolerant strain. Effect of different ratios of two strains was investigated and xylose-utilizing strain to inhibitor-tolerant strain ratio of 10: 1 (w/w) showed the best xylose consumption and the highest ethanol yield. Inoculating of xylose-utilizing strain at the later stage of SSCF (24-48 h) exhibited lower ethanol yield than inoculating at early stage (the beginning 0-12 h), probably due to the reduced enzymatic efficiency caused by the unconsumed xylose and oligomeric sugars. Co-culture SSCF increased ethanol concentration by 21.2% and 41.0% comparing to SSCF using individual inhibitor-tolerant and xylose-utilizing strain (increased from 48.5 and 41.7 g/L to 58.8 g/L), respectively, which suggest this co-culture system was very promising. (C) 2016 Elsevier Ltd. All rights reserved.