D-Allose is a rare cis-caprose with a wide range of physiological functions, which has a wide range of applications in medicine, food, and other industries. L-Rhamnose isomerase (L-Rhi) is the earliest enzyme found to catalyze the production of D-allose from D-psicose. This catalyst has a high conversion rate, but its specificity for substrates is limited; thus, it cannot fulfill the requirements of industrial production of D-allose.In this study, L-Rhi derived from Bacillus subtilis was employed as the research subject, and D-psicose as the conversion substrate. Two mutant libraries were constructed through alanine scanning, saturation mutation, and rational design based on the analysis of the secondary structure, tertiary structure, and interactions with ligands of the enzyme. The yield of D-allose produced by these mutants was assessed; it was found that the conversion rate of mutant D325M to D-allose was increased by 55.73 %, and the D325S improved by 15.34 %, while mutant W184H increased by 10.37 % at 55 °C, respectively.According to modeling analysis, manganese (Mn2+) had no significant effect on the production of D-psicose from D-psicose by L-Rhi. The results of molecular dynamics simulation demonstrated that the mutants W184H, D325M, and D325S had more stable protein structures while binding with the substrate D-psicose, as evidenced by its root mean square deviation (RMSD), root mean square fluctuation (RMSF), and binding free energy values. It was more conducive to binding D-psicose and facilitating its conversion to D-allose, providing the basis for the production of D-allose.