Ethanol can be produced from lignocellulosic biomass with the usage of ball milling pretreatment followed by enzymatic hydrolysis and fermentation. The sugar yields from lignocellulosic feed stocks are critical parameters for ethanol production process. The research results from this paper indicated that the yields of glucose and xylose were improved by adding any of the following dilute chemical reagents: H2SO4, HCl, HNO3, CH3COOH, HCOOH, H3PO4, and NaOH, KOH, Ca(OH)(2), NH3 center dot H2O in the ball milling pretreatment of corn stover. The optimal enzymatic hydrolysis efficiencies were obtained under the conditions of ball milling in the alkali medium that was due to delignification. The data also demonstrated that ball milling pretreatment was a robust process. From the microscope image of ball milling-pretreated corn stover, it could be observed that the particle size of material was decreased and the fiber structure was more loosely organized. Meanwhile, the results indicate that the treatment effect of wet milling is better than that of dry milling. The optimum parameters for the milling process were ball speed of 350 r/min, solid/liquid ratio of 1:10, raw material particle size with 0.5 mm, and number of balls of 20 (steel ball, I broken vertical bar = 10 mm), grinding for 30 min. In comparison with water milling process, alkaline milling treatment could increase the enzymatic hydrolysis efficiency of corn stover by 110%; and through the digestion process with the combination of xylanase and cellulase mixture, the hydrolysis efficiency could increase by 160%.