Lactulose holds broad application due to its unique pharmaceutical and prebiotic properties. Cellobiose 2-epimerase (CE) efficiently catalyzes the production of lactulose, offering an eco-friendly biosynthetic alternative. However, its relatively low isomerization activity hampers widespread application in the manufacture of lactulose. In this study, we selected DtCE sourced from Dictyoglomus thermophilum and successfully generated mutants M4 and M5 through an innovative loop engineering process that integrates computer-aided design with directed evolution. Remarkably, the isomerization activities of the mutants M4 and M5 increased by 46.5 % and 81.8 %, respectively, marking a significant leap forward compared to the wild type. Conversely, their epimerization activities underwent a dramatic decline, dropping by 80.0 % and 50.0 %, respectively. Therefore, these mutants demonstrated considerable superiority in the synthesis of lactulose. Moreover, molecular dynamics simulations showed that modifications of the flexible loop affected protonation, enhancing isomerization selectivity. This study underscores the precision and efficacy of our engineering approach in customizing DtCE's properties to meet specific needs, while concurrently establishing a technical foundation for industrial-scale biosynthetic production of lactulose.