D-Allulose, an important rare sugar, has a wide range of application value in food, cosmetics and pharmaceutical industry. At present, biological methods are mainly used for D-allulose production in industry. Due to the disadvantages of the traditional enzymatic methods, such as complicated steps, high cost and difficult product separation and purification, it has been difficult to meet the needs of industrial production. In recent years, whole-cell synthesis systems have attracted much attention because of their low cost, convenient operation and easy separation. The study is aimed at heterologously expressing D-allulose-3-epimease (DAEase) from Caballeronia insecticola in Bacillus subtilis WB800 to catalyze D-fructose to D-allulose. Recombinant strains with different constitutive promoters were firstly designed and constructed to improve the expression of DAEase in B. subtilis. Then, the conditions of the whole cell reaction system (temperature, pH, metal ions, cell concentration) were optimized, and the conversion efficiency of D-fructose under different substrate concentrations was explored. The results showed that the promoter PylbP could maximally express DAEase in B. subtilis WB800, and the optimal temperature, pH and metal ions of recombinant DAEase were 65℃, pH 9.5 and 5 mmol/L Mg2+, respectively. The whole cell reaction almost reached to equilibrium in 4 h with a conversion rate of 30.06% when 500 g/L D-fructose was added as the substrate. This research provided experimental and theoretical basis for producing D-allulose industrially.