Recombinant Escherichia coli whole cells harboring Bacillus licheniformis L-arabinose isomerase (BLAI) were immobilized with alginate. The operational conditions for immobilization were optimized with response surface methodology. Optimal alginate concentration, Ca(2+) concentration, and cell mass loading were 1.8% (w/v), 0.1 M, and 44.5 g L(-1), respectively. The interactions between Ca(2+) concentration, alginate concentration, and initial cell mass were significant. After immobilization of BLAI, cross-linking with 0.1% glutaraldehyde significantly reduced cell leakage. The half-life of immobilized whole cells was 150 days, which was 50-fold longer than that of free cells. In seven repeated batches for L-ribulose production, the productivity was as high as 56.7 g L(-1) h(-1) at 400 g L(-1) substrate concentration. The immobilized cells retained 89% of the initial yield after 33 days of reaction. Immobilization of whole cells harboring BLAI, therefore, makes a suitable biocatalyst for the production of L-ribulose, particularly because of its high stability and low cost.