L-Ribulose and L-ribose are two high-value unnatural sugars that can be biosynthesized by sugar isomerases. In this paper, an L-arabinose isomerase (BvAI) fromBacillus velezensisCICC 24777 was cloned and overexpressed inEscherichia coliBL21 (DE3) strain. The maximum activity of recombinant BvAI was observed at 45 degrees C and pH 8.0, in the presence of 1.0 mM Mn2+. Approximately 207.2 g/L L-ribulose was obtained from 300 g/L L-arabinose in 1.5 h byE. coliharboring BvAI. In addition, approximately 74.25 g/L L-ribose was produced from 300 g/L L-arabinose in 7 h byE. colico-expressing BvAI and L-RI fromActinotalea fermentansATCC 43279 (AfRI). This study provides a feasible approach for producing L-ribose from L-arabinose using a co-expression system harboring L-Al and L-RI.