Abstract: : L-Arabinose is a crucial component of hemicellulose hydrolysate. In the chemical production process of xylitol, it is often discharged along with the xylose mother liquor, leading to resource waste and environmental pollution. In this study, a biosynthetic pathway for converting L-Arabinose into xylitol was established in Escherichia coli W3110. A multicistron expression module containing three enzymes required for xylitol biosynthesis, namely DPE, AraA, and LXR, was constructed. Meanwhile, the araBAD and lyxK genes in the L-Arabinose metabolic branch pathway of the E. coli W3110 genome were knocked out, and the xylitol- producing strain ∆W-DLA was successfully obtained. Shake-flask fermentation was carried out using 10 g·L⁻¹, L-Arabinose as the substrate and 10 g·L⁻¹ glycerol as the co-substrate. The results showed that after 30 h of fermentation, the xylitol yield reached 8.26 g·L⁻¹, and the conversion rate of L-Arabinose was 92.3%. The culture medium of the engineered strain ΔW-DLA was optimized through single-factor experiments and response-surface experiments. After optimization, ∆W-DLA produced 9.46 g·L⁻¹ xylitol after 24 hours of fermentation, and the xylitol production rate was increased by 39.3% compared with that before optimization. This study provides a new way for the resource-utilization of xylose mother liquor.