Abstract: To develop a novel D-psicose-3-epimerase possessing higher thermal stability, an exploration was carried out on the genome of the intestinal probiotic Blautia wexlerae. Subsequently, the suspected D-psicose-3-epimerase gene was expressed in Bacillus subtilis. The function of the expressed target protein was determined by high-performance liquid chromatography. Rational design strategies were implemented to enhance the enzyme's thermal stability. The findings revealed that the sugar phosphate isomerase gene of B. wexlerae exhibited the enzyme activity of D-psicose-3-epimerase (DPE). The optimal temperature and pH for enzyme activity were 60 ℃ and 7.0, and the conversion yield was 27.1%±1.1%. Notably, the mutant DPE (H283A) demonstrated an optimal temperature that was 5 ℃ higher than that of the wild type. Its half-life at 70 ℃ was 30 minutes, which was 10 min extended compared to the wild type DPE (WT). Moreover, the conversion yield achieved was 30.6%±2.6%, signifying a 3.5% improvement over DPE (WT). This research successfully mined and identified the D-psicose-3-epimerase of B. wexlerae, and acquired the mutant DPE (H283A), which exhibited enhanced thermal stability and enzyme activity through rational design.