D-Ribulose, a potentially valuable rare sugar and an excellent building block in asymmetric synthesis, is usually produced by chemical synthesis, which unfortunately does not meet the increasing demand. In addition, ribitol oxidization for the production of d-ribulose has not yet become an industrial reality because of the lack of efficient biocatalysts, resulting in higher production costs as well as a poor yield. In this study, we have employed the Enterobacter aerogenes ribitol: NAD⁺ 2-oxidoreductase (EaRDH), which could efficiently and selectively convert ribitol to D-ribulose. The purified EaRDH enzyme and the recombinant Escherichia coli strain (as a whole-cell catalyst) were used to produce d-ribulose. Ribitol was efficiently converted to d-ribulose by EaRDH with a yield of ~85%, suggesting the usefulness of this enzyme for the in vivo and in vitro production of d-ribulose from ribitol. The oxidation of ribitol to D-ribulose by EaRDH was accomplished in the presence of stoichiometric amounts of NAD⁺; therefore, D-ribulose production was further enhanced by the incorporation of diaphorase for continuous NAD⁺ regeneration. The biocatalytic process presented should be a promising route for the biotechnological production of d-ribulose (and related branched pentoses) at an efficient and industrially relevant scale.