Ethylenediamine (EDA) pretreatment is an effective pretreatment technology to improve enzymatic digestibility of corn stover for the production of fermentable sugars. In this study, key pretreatment parameters were identified and optimized to improve enzymatic digestibility of corn stover. We found that agitation and biomass stack height during pretreatment had significant impacts on enzymatic digestibility. Response surface experiment showed that optimal condition to achieve maximum total sugar enzymatic yield was 150<SUP>o</SUP>C and 80mL EDA/100g corn stover. Under this condition, glucose yield was greater than 90% in enzymatic hydrolysis at 1% glucan loading. Optimized temperature to minimize residual EDA in pretreated corn stover was 200<SUP>o</SUP>C. Two-stage pretreatment was carried out to maximize both sugar yields and EDA removal, in which glucose and xylose enzymatic yields reached 92% and 70% respectively at 1% glucan loading, and EDA residue reduced to 27g/kg corn stover. With the optimal enzyme loadings (both enzymes of Ctec2 and Htec2 were loaded at 30mg protein/g glucan), glucose and xylose yields at 6% glucan loading reached 81% and 58%, respectively.