D-Allulose, an important rare sugar, has a wide range of application value in food, cosmetics and pharmaceutical industry. At present, D-allulose production methods in industry are mainly biological methods. Due to the disadvantages of the traditional enzymatic methods, such as complicated steps, high cost and difficulty in product separation and purification, it was 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 was aimed at efficient heterologously expressing of D-allulose 3-epimease (DAEase) originated from Caballeronia insecticola in production safety strains Bacillus subtilis (B. 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 PylbP could maximally express recombinant DAEase in B. subtilis WB800, and the optimal temperature, pH and metal ions of recombinant DAEase in whole cell reaction system were 65℃, 9.5 and 5mmol/L Mg²⁺, respectively. The whole cell reaction could almost reach to equilibrium in 4 h with a conversion rate of 30.06% when 500g/L D-fructose was added as substrate. This research provided experimental and theoretical basis for producing D-allulose industrially.