Background and Objective: Corncob contain high percentage of cellulose (40.95±0.38%), hemicellulose (38.94±1.72%) and low lignin content (16.54±0.38%) which has a great potential of bioconversion to bioethanol. The objective of this research was to evaluate the effect of acidification, freezing and the combination of these pre-treatments in corncob compounds and its enzymatic hydrolysis. Materials and Methods: It was evaluated the changes occured in macrostructure and lignocellulosic compounds after pre-treatments. Corncob fiber was pretreated by freezing at -20°C for 12 h; with 0.25 M sulfuric acid, pH 2.0, at 121°C for 20 min and by the combination of both pre-treatments. After pre-treatment, an enzymatic hydrolysis with 150 Ucellulase/gsubstrate in 10 mL of acetate was performed at 100 rpm, 37°C and pH 5.48 for 72 h. Data were analyzed by one-way analysis of variance using SPSS. Results: The combined pre-treatment (acidification and freezing) allowed to obtain the highest amount of cellulose (64.12±1.03%) and lignin (19.76±0.42%) and low amount of hemicellulose ( 10.16±1.42%) was determined. The optical microscopy analysis of samples with combined pre-treatment show partitioned and less agglomerated structure. Conclusion: The higher concentration of glucose (40.90±2.11 g LG1 ) was obtained by combined pre-treatment, which increased the enzyme efficiency with 57.41%, which could be approved for the conversion of high value products, like bioethanol.