Background The production of second generation ethanol from lignocellulosic biomasses that have not had their potential fully explored as feedstock is of great importance. Arundo donax is one these biomasses. It is a promising grassy plant to be used as a renewable resource for the production of fuels and chemicals, because of its fast growth rate, ability to grow in different soil types and climatic conditions. The present study evaluated its use as feedstock for the production of second generation ethanol. Results Initially its chemical characterization was carried out, and a protocol for fractioning the biomass through diluted acid pretreatment followed by alkaline pretreatment was developed, providing a solid fraction which was undergone to enzymatic hydrolysis reaching 42 g/L of glucose, obtained in 30 h of enzymatic hydrolysis. This partially delignified material was subjected to a simultaneous saccharification and fermentation (SSF) process, resulting in an ethanol concentration of 39 g/L at 70 h. Conclusions The fermentability of the pretreated biomass was performed successfully through the conception of simultaneous saccharification and fermentation resulting in approximately 75 L of ethanol per ton of cellulose.