A novel two-step process for pretreatment of corn stover was investigated with the goal of increasing sugar recovery and decreasing the capital cost. In the process, corn stover was first treated with dilute hydrochloric acid to maximize xylan recovery, and then the residue was treated with aqueous ammonia to alter the lignin structure and swell the cellulose surface. The optimal conditions were 110 °C and 40 min for 1% dilute hydrochloric acid pretreatment with a liquid to solid ratio of 10:1 followed by aqueous ammonia pretreatment at 37% NH3, 130 °C, 30 min, and liquid to solid ratio of 6:1. Under these pretreatment conditions, the glucan and xylan recoveries were 83.2% and 97.3%, respectively, with cellulase dosage at 15 FPU/g of substrate. When the cellulase dosage was decreased from 15 FPU/g to 10 FPU/g of substrate, the glucan recovery only dropped to 70%, while the xylan recovery remained high at 95.1%. The results show that this two-step pretreatment was effective for achieving high sugar recovery from corn stover by enzymatic hydrolysis.