In this study, DEAE-cotton [derivatized by 2-(diethylamino)ethyl chloride hydrochloride (DEAE·HCl)] was prepared as a carrier for immobilized Pichia stipitis for ethanol production. When cotton was derivatized with 0.5 M DEAE·HCl, the yeast cell suspension was adsorbed at 100% of the initial cell OD600. The adsorbed yeast cells were estimated to be 101.8 mg-dry cells/g-DEAE-cotton. In particular, when a flask culture using the immobilized yeast cells was conducted in a glucose and xylose-containing medium, the yeast cells on the DEAE-cotton gradually produced ethanol, according to glucose and xylose consumption; the ethanol yield was approximately 0.33 g-ethanol/g-monosaccharide. Because DEAE-cotton was successfully used as a carrier for ethanol production from a glucose and xylose-containing medium, we expect that this bioethanol production process may be used for the bioethanol production process from the hydrolysate of lignocellulosic biomass. All the results of DEAE-cotton were compared with those of DEAE-cellulose as a carrier for immobilization.