Cellobiohydrolase, a workhorse enzyme for cellulose degradation, has the potential for application in biofuels, biochemicals, biomaterials, etc. However, highly efficient cellobiohydrolase is desirable to improve the economic feasibility of industrial applications. In this work, two variants TrCel7AT97A and TrCel7AK166A were designed to increase the flexibility of the entrance of the catalytic tunnel. We performed the molecular dynamics simulations using NAMD for 100 ns at 300 K in the NPT (isothermal–isobaric) ensemble and then we found that the region flexibility at the catalytic tunnel entrances of TrCel7AT97A and TrCel7AK166A was improved, compared to that of TrCel7A, based on the calculation of the average root mean square fluctuation. The specific activities of TrCel7AT97A and TrCel7AK166A were increased by 37 % and 68 %, respectively, compared with that of TrCel7A at 45 °C for 12 h. An effective approach named revive amino acid flexibility for enzyme activity based on amino acid flexibility was proposed for protein engineering of cellobiohydrolase TrCel7A.