The economics driving biorefinery development requires high value-added products such as cellobiose for financial feasibility. This research describes a simple technology for increasing cellobiose yields during lignocellulosic hydrolysis. The yield of cellobiose produced during cellulose hydrolysis was maximized by modification of reaction conditions. The addition of an inhibitor from the group that includes glucose oxidase, gluconolactone, and gluconic acid during cellulase hydrolysis of cellulose increased the amount of cellobiose produced. The optimal conditions for cellobiose production were determined for four factors; reaction time, cellulase concentration, cellulose concentration, and inhibitor concentration using a Box-Behnken experimental design. Gluconolactone in the cellulase system resulted in the greatest production of cellobiose (31.2%) from cellulose. The yield of cellobiose was 23.7% with glucose oxidase, similar to 21.9% with gluconic acid.