We have developed a whole-cell bioconversion system for the production of D-1,2,4-butanetriol (BT) from renewable biomass. A plasmid pETduet-xylB-yjhG-T7-adhP-T7-mdlC was constructed and transformed to Escherichia coli BL21(DE3) to obtain the whole cells of E. coli BL21-XYMA capable of bioconversion D-xylose to BT. Then, the factors including carbon sources, nitrogen sources, metal ions, and culture conditions (pH, temperature, IPTG) were identified, and their effects on the whole-cell activity for BT production were investigated. To obtain the highest whole-cell activity, the optimal cultivation parameters are: 15 g·L⁻¹ yeast extract, 5 g·L⁻¹ sucrose, 3 g·L⁻¹ KH₂PO₄, 5 g·L⁻¹ NaCl, 3 g·L⁻¹ NH₄Cl, 0.25 g·L⁻¹ MgSO₄∙7H₂O and 1 mL·L⁻¹ the mixture of trace elements. With the optimized whole cells of E. coli BL21-XYMA, 60 g·L⁻¹ of xylose was converted to 28 g·L⁻¹ BT with a molar yield of 66.0%, which is higher than those reported in the biotechnological system.