D-ribose, a five-carbon sugar with diverse applications, is mainly produced by transketolase(tkt)-deficient Bacillus subtilis (B. Subtilis). We constructed B.subtilis SFR-3A by replacing the corresponding sites of B. subtilis 168 with the mutation site of tkt in the “wild” D-ribose high-producing strain B. subtilis SFR-4, resulting in 5.29 g/L of Dribose. In the meantime, B.subtilis SFR-159 was constructed by completely removing the tkt gene of B. subtilis 168 with a higher production of 6.21 g/L. Through medium optimization, batch fermentation of SFR-3A and SFR-159 gave the best result of 27.56 g/L and 29.89 g/L, which corresponds to productivity of 0.51 g/L/h and 0.41 g/L/h, respectively. In this work, SFR-3A showed more latent capacity over SFR-159 as to productivity and had greater potential to serve as a platform for D-ribose production.