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Due to the high potential of the extrusion technique for pretreatment of lignocellulosic substrates, several attempts have been conducted in previous studies to further increase the subsequent sugar yield from extrusion pretreatment. Examples include application of chemicals along with extrusion, such as alkali-extrusion and ethylene glycol-extrusion, or after extrusion, such as hot water extraction. In this study a new sequential technique has been developed for pretreatment of corn stover, which utilizes an initial extrusion pretreatment at the previously optimized extrusion of 90 C, 180 C, and 180 C corresponding to feed zone, barrel zone and die zone, respectively, followed by pretreatment with poly ethylene glycol 6000 (PEG) to prevent non-productive adsorption of enzyme to the substrate. In order to fully characterize the response for sugar yield over the range of surfactant treatment conditions assessed, response surface methodology was used. Treatment temperature, incubation time and PEG concentration were varied between 45-55C, 1-4 h, 0.15-0.6 g PEG/g glucan, respectively. Statistical analysis was conducted by fitting the data to a quadratic polynomial model. PEG concentration and temperature were found to be the most significant factors and not independent from one another. The optimum condition (using Ctec and Htec enzymes) resulted in 25.4% and 10.3% increase in glucose and xylose yield, respectively. Using the combination of 12% g Ctec2/ g glucan and 0.3 g PEG/ g glucan, the glucose yield of extruded CS reached 98%. Adding additional PEG during hydrolysis did not facilitate further increase of the sugar yield. Decreased adsorption of enzyme to the lignocellulosic substrate as a result of kinetic parameters evaluation and nitrogen combustion analysis suggested an increased solubilization of cellulase in the presence of PEG. We suggest that a non-productive adsorption of enzymes occur during hydrolysis not only due to lignin but also due to crystalline cellulose.