Cyclodextrin is used widely in industry, and the thermostability of cyclodextrin glucanotransferase (CGTase) is important for high-yield cyclodextrin production. A chemical mixture (25% glycerol, 10% polyethylene glycol 400, and 0.5 mmol/L CaCl2) was used as stabilizer to enhance the thermostability of CGTase after optimizing its concentration, which resulted in a synergistic effect on CGTase stability. The beta-CGTase and gamma-CGTase activities increased by 7.6- and 9.4-fold, respectively, compared with controls, which resulted from an increase in the energy of inactivation, as determined by a thermokinetic analysis. The optimum temperature of CGTase activity increased from 50 to 60 degrees C after the stabilizer application. The stabilizer was applied to cyclodextrin production, and it resulted in enhanced CGTase activity, which showed higher increases of starch to CD conversion at 60 and 90 degrees C. These results demonstrate that using a stabilizer is an effective approach for CGTase storage and cyclodextrin production.