Conversion of lignocellulose into fermentable sugars and other chemicals usually requires multi-step unit operations, such as pretreatment, filtration/washing, and enzymatic saccharification and fermentation, which are the core steps responsible for increased operating expenses. A low-temperature NaOH/urea solution was shown to be an efficient cellulose solvent for overcoming the recalcitrance of lignocellulose by partially or fully converting rigid cellulose I crystallite into the more easily digestible cellulose II structure and by extracting a majority portion of the lignin and xylan from the lignocellulose. Higher yields of fermentable sugars were produced directly from corn stover in one vessel. This one-pot production of fermentable sugars was achieved via a combination process, including pretreatment with low-temperature NaOH/urea solution, pH adjustment, and enzymatic saccharification in a single reactor. This one-pot process liberated 86.3% of glucose and 71.3% of xylose in 24 h at an enzyme loading of 10 FPU/g and solid loading of 5%. Surfactant addition further enhanced enzymatic saccharification. The combination of low-temperature NaOH/urea pretreatment and enzymatic saccharification into a one-pot process is an efficient method for the conversion of lignocellulose into fermentable sugars suitable for conversion into fuels and other chemicals. Further studies related to lignin recovery and economical evaluation will be conducted.