A rational design method to improve beta-mannanase (ManTJ102) thermostability was developed successfully in this study. The flexible area of residues 330-340 in ManTJ102 was firstly selected from analysis of molecular dynamics simulation and then the critical amino acid residue (Ala336Pro) with the lowest mutation energy was determined by virtual mutation, whose mutant was named as Mutant336. Afterward, the dynamics transition temperature (T-dtt) of ManTJ102 and Mutant336 was evaluated by simulated annealing and heating, and Mutant336 with higher T-dtt was implemented for experimental verification of the enzyme thermostability. As a result, the half-life of Mutant336 activity was 120 min at 60 degrees C, which was 24-fold of ManTJ102, and the irreversible thermal denaturation constant of Mutant336 was only about 2/5 of ManTJ102, indicating that Mutant336 has better thermostability than ManTJ102. Furthermore, Mutant336 has much higher beta-mannanase activity and specific activity than ManTJ102. Therefore, Mutant336 was more suitable to further research for applications.