Rapeseed straw, containing about 50% of carbohydrates was used as a low-cost raw material for bioethanol production. Since xylose represents about 30% of total sugars in this feedstock, the use of microorganisms capable of metabolizing both pentoses and hexoses is crucial. In this work, rapeseed straw was pretreated with sulfuric acid at previously determined optimal conditions. Then, the whole slurry obtained after pretreatment was enzymatically hydrolyzed and the resulting sugar solution was cofermented by Scheffersomyces stipitis CBS654 and Escherichia coli MM160. Prior to fermentation, different detoxification methods were tested in order to increase the fermentability of the hydrolysate. Only the detoxification by ion-exchange resins achieved a fermentable hydrolysate due to the almost complete removal of phenolics and acetic acid as well as high reduction in levels of furans and formic acid. The best results of ethanol production corresponded to the fermentation of rapeseed straw hydrolysate with E. coli after detoxification by ion-exchange resins. In this case, an ethanol concentration of 40 g/L was reached that corresponds to 85% of the theoretical ethanol yield. The use of this new process configuration for rapeseed straw by enzymatic saccharification of the whole slurry and subsequent separation allowed the fermentation of all sugars in a single stage, avoiding the problems associated with the presence of solids in the fermentation.