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The present study was designed to find a suitable microaerobic condition and ratio of glucose and xylose for maximum ethanol production using co-culture of Saccharomyces cerevisiae and Pichia stipitis. The maximum ethanol concentration and yield were achieved at 0.05 vvm aeration rate and 2:1 glucose/xylose ratio. The co-culture resulted in maximum ethanol concentration, ethanol yield, and volumetric productivity of 12.33 ± 0.10 g/L, 0.43 g/g, and 0.26 g/L/h, respectively. While, the monoculture of P. stipitis resulted in 8.96 ± 0.13 g/L, 0.36 g/g, and 0.19 g/L/h respectively. The fermentation carried out in microaerobic mode delivered 10.68% and 10.56% more ethanol concentration and ethanol yield respectively from glucose compared to the combination of anaerobic and microaerobic mode. Also, the glucose uptake rate increased to 0.83 g/L/h, which corresponds to an improvement of 50.16%, suggesting that the lower microaerophilic condition not only supports P. stipitis metabolism but also does S. cerevisiae to convert glucose faster in a co-culture system. Hence, co-culture cultivation in microaerobic mode would be a better condition to achieve maximum ethanol and productivity. [ABSTRACT FROM AUTHOR]