A protoplast fusion between S. cerevisiae and xylose-fermenting yeast Pichia stipitis ATCC 58785 was conducted to produce the fusant which could improve bioethanol production. Among the five selected fusants, the fusant F24 (MH429783.1) was able to attain maximum bioethanol production from the glucose-xylose mixture. The fusant F24 produced 30.24 g/L bioethanol. Bioethanol has been gaining attention as a clean and renewable fuel because of its immense environmental benefits. Worldwide bioethanol demand has been continuously increasing due to the rapid growth in population and industrialization. Co-fermentation was achieved by P.stipitis ATCC 58785 in the medium containing the glucose and xylose mixture. P.stipitis ATCC 58785 fermented both sugars and produced 28.30 g/L bioethanol. The co-culture fermentation of S. cerevisiae and P. stipitis ATCC 58785 produced 30.12 g/L bioethanol. The fermentation using fusant gives the advantage to the production of bioethanol by reducing the duration of fermentation and cost of production.