Cellobiose has biotechnological potential for value-added products in food, cosmetic, and pharmaceutical industries. This work utilized experimental design methodology to improve cellobiose production from cellulose by cellulase in the presence of gluconolactone. Preliminary tests were employed to obtain the favorable conditions for cellulase activity analysis and found that the optimal temperature and pH were 37°C and 5.2, respectively. Moreover, the inhibition pattern of gluconolactone on cellulase was investigated. The optimal conditions for cellobiose production were determined by response surface methodology (RSM). A three-level design of 29 experiments was conducted with four factors, each of which was designed to study reaction time, cellulose concentration, cellulase concentration, and gluconolactone concentration. Our statistical model predicted that the highest yield of cellobiose would be 9.4 g/L under the following optimized reaction condition of 1.5% of cellulose, cellulase 3.8 FPU, 3.0% of gluconolactone, and 20 hr reaction time.