This work provides the suitability of using near-critical CO2 to assist the enzymatic hydrolysis of starch from brewer's spent grain (BSG). Total reducing sugars (TRS) were obtained in different conditions of temperature (30 degrees C-50 degrees C), pressure (100 bar-250 bar) and moisture (40%-80%) using a central composite rotatable design. The concentration of enzyme in relation to substrate (BSG) was maintained constant at 8.2 wt.%. The highest TRS yield (219.39 g/kg) was reached with enzyme-substrate mixture and CO2 at 40 degrees C, 175 bar, and 80% moisture for 240 min. In this condition, approximately 26.5 wt.% glucose, 2.2 wt.% cellobiose, 1.7 wt.% xylose and 0.7 wt.% arabinose were quantified in the hydrolyzed medium. Using near-critical CO2 increased the sugars yield by 3.6 times higher than that of conventional enzymatic hydrolysis.