The growing interest on rape oil as raw material for biodiesel production has resulted in an increasing availability of rape straw, an agricultural residue that is an attractive renewable source for the production of second-generation bioethanol. Pretreatment is one of the key steps in such a conversion process. In this work, a sequential two-stage pretreatment with dilute sulfuric acid (130 °C, 60 min, 2% w/v H2SO4) followed by H2O2 (1-5% w/v) in alkaline medium (NaOH) at low temperature (60, 90 °C) and at different pretreatment times (30-90 min) was investigated. The first-acid stage allows the solubilisation of hemicellulose fraction into fermentable sugars. The second-alkaline peroxide stage allows the delignification of the solid material whilst the cellulose remaining in rape straw turned highly digestible by cellulases. Simultaneous saccharification and fermentation with 15% (w/v) delignified substrate at 90 °C, 5% H2O2 for 60 min, led to a maximum ethanol production of 53 g/L and a yield of 85% of the theoretical.