Trichoderma reesei endoglucanases (EGs) have limited industrial applications due to its low thermostability and activity. Here, we aimed to improve the thermostability of EGs from T.reesei without reducing its activity counteracting the activity-stability trade-off. A cross-strategy combination of machine learning and B-factor analysis was used to predict beneficial amino acid substitution in EG loop optimization. Experimental validation showed single-site mutated EG concomitantly improved enzymatic activity and thermal properties by 17.21%–18.06% and 49.85%–62.90%, respectively, compared with wild-type EGs. Furthermore, the mechanism explained mutant variants had lower RMSD values and a more stable overall structure than the wild type. According to this study, EGs loop optimization is crucial for balancing the activity-stability trade-off, which may provide new insights into how loop region function interacts with enzymatic characteristics. Moreover, the cross-strategy between machine learning and B-factor analysis improved superior enzyme activity-stability performance, which integrated structure-dependent and sequence-dependent information.