Background: The technology of converting corn mashes to ethanol has been mature, but corn mashes has high-viscosity and high-sugar characteristics which hindered cellulose utilization and yeast-fermentation efficiency. The excessive viscosity of corn mash is caused by the presence of non-starch polysaccharides, such as cellulose in cereal grains. Corn kernel fiber (mostly cellulose) is typically unconverted in the process. Results: A novel lignocellulolytic enzymes cocktail with strong substrate specificity was prepared for high-viscosity, high-sugar corn mash. The in situ conversion of corn mashes with novel lignocellulolytic enzymes at the optimum cellulase dosage of 50 FPU/L resulted in 12.4%, 12.0%, 11.8%, and 12.9% increased ethanol concentration compared with the reference mash at 0.3, 1, 5, and 70 L batch-fermentation scales, respectively. The highest yield of ethanol from corn mash digested with the prepared novel lignocellulolytic enzyme reached 117.0 ± 0.1g/L at the 70 L batch fermentation, which was a 12.9% increase in ethanol yield. Adding the lignocellulolytic enzymes caused the greatest decrease in viscosity of corn mash by 40.9% compared with the reference mash (33.5 ± 1.5 Pa·s), whereas the residual sugars decreased by 56.3%. Simultaneously, the application of novel lignocellulolytic enzymes increased the value of dried distiller’s grain with solubles by increasing the protein content and decreasing the residual cellulose and starch content.Conclusion: The application of novel lignocellulolytic enzymes significantly improved the alcohol concentration, productivity, and yield. With the same amount of material, the application of the novel enzymes cocktail can enhance the ethanol yield by more than 10%. The in situ conversion of cellulose promoted the release of contents, including starch and protein, which can decrease the fermentation broth viscosity and improve the rheological property, thereby improving the ethanol yield. Thus, this technology can increase the net revenue of fuel-ethanol industrialization and promote the technological progress of renewable energy.