Abstract: Lignocellulosic biomass, particularly agricultural residues such as corn stalks, represents a vast and underutilized resource for sustainable biofuel and biochemical production. However, the recalcitrance of lignocellulose, especially under high-solids conditions, poses significant challenges in terms of enzymatic hydrolysis efficiency, mass transfer limitations, and product inhibition. This study aims to address these bottlenecks by optimizing the enzymatic saccharification of holocellulose derived from corn stalks using a tailored enzyme cocktail composed of cellulase, cellobiase, and xylanase. The objective was to enhance sugar yield and concentration under industrially relevant high-solids loadings.Corn stalk holocellulose, supplied by Anhui Fengyuan Group, was pretreated using ammonium sulfate-based high-temperature steam explosion, resulting in a substrate containing 40.37% cellulose, 11.00% hemicellulose, 9.80% lignin, and 18.81% ash. The enzymatic hydrolysis process was systematically optimized using a combination of single-factor experiments and Box-Behnken Design (BBD) coupled with Response Surface Methodology (RSM). Four key variables—substrate solid content (15–25%, w/w), enzyme dosage (2–4×), temperature (45–55 ℃), and residence time (24–72 h)—were evaluated for their individual and interactive effects on reducing sugar yield.The optimized conditions were determined to be 20% solid content, 3× enzyme dosage (equivalent to 27.71 mL cellulase, 17.08 mL cellobiase, and 1.14 mL xylanase), 48.21 ℃, and a hydrolysis duration of 72 h. Under these conditions, the reducing sugar concentration reached 108.97 g/L, corresponding to a yield of 94.49%, which represented a 21.3% improvement over the baseline process. The model exhibited high reliability, with a coefficient of determination (R²) of 0.9157 and an adjusted R² of 0.8315, indicating a strong fit between experimental and predicted values. Structural characterization of the substrate before and after enzymatic hydrolysis was conducted using SEM, FTIR, XRD, BET, and zeta potential analysis. SEM images revealed increased surface porosity and disruption of the intact cell wall structure following enzymatic treatment. FTIR spectra confirmed the degradation of hemicellulose and partial modification of lignin structure, as evidenced by the reduction or disappearance of characteristic acetyl and aromatic peaks. XRD analysis showed a notable decrease in cellulose crystallinity index (CrI) from 13.91% to 9.43% after 24 h of hydrolysis, indicating effective disruption of crystalline cellulose regions. BET surface area analysis demonstrated a reduction in total surface area but an increase in micropore volume and average pore diameter, suggesting enhanced accessibility of enzyme to internal cellulose structures. Zeta potential and particle size measurements indicated a reduction in particle aggregation and improved dispersion stability, further facilitating enzymatic action.These findings underscore the effectiveness of multi-enzyme synergy in overcoming the structural and physicochemical barriers of lignocellulosic biomass under high-solids conditions. The study provides a scalable and efficient enzymatic hydrolysis strategy that significantly increases sugar concentration and yield, offering a promising pathway for the industrial valorization of corn stalks and similar lignocellulosic feedstocks. Future work should focus on enzyme engineering for improved thermal stability and resistance to lignin-induced deactivation, as well as the integration of process engineering solutions such as fed-batch or continuous systems to further enhance economic viability and environmental sustainability.