L-Xylulose is a rare ketose with potential applications in the pharmaceutical and food industries. This metabolic intermediate can be synthesized by xylitol 4-dehydrogenase (XDH) using xylitol as substrate and NAD⁺ as a cofactor. A novel XDH from Erwinia aphidicola SK47.001 was expressed in food-grade Bacillus subtilis and biochemically characterized. The purified enzyme exhibited an optimal temperature of 45 °C, an optimal pH of 11, a K_m of 18.71 mM, and a V_max of 0.52 μmol/(min mg). To improve XDH activity, another B. subtilis strain was constructed to co-express XDH and the water-forming enzyme NADH oxidase (NOX), an efficient recycler of NAD⁺. In this case, the XDH activity was 3.4-fold higher than that of the single enzyme. The putative structure and probable catalytic active sites of XDH were explored by homology modeling and molecular docking. The amino acids Ser149, Tyr162, and Lys166 were identified as potential XDH catalytic active sites for xylitol and NAD⁺.