Corn stalk was pretreated by aqueous tetrabutylammonium hydroxide to enhance enzymatic hydrolysis process. The effects of temperature (20–80 °C), concentration of tetrabutylammonium hydroxide (10–40 wt%), time (5–60 min) and ratio of solid to liquid (1:4–1:8, wt/wt) were investigated to find the optimal operational condition, which was determined as 20 °C, 30 wt%, 20 min and 1:5 respectively. This was a quite mild pretreatment condition resulting in 68.17% enzymatic hydrolysis sugar yield and the removal rate of hemicelluloses and lignin were 23.5% and 31.5% respectively. Besides, the microstructure and crystal structure changes of raw material and pretreated samples were analyzed by scanning electron microscope and X-ray diffraction respectively. Finally, a possible mechanism of pretreatment process was put forward based on the comprehensive analysis.