L-Ribose and L-arabinose were prepared biochemically from ribitol via a two-step reaction, by which the complete oxidation of ribitol to L-ribulose (similar to 98%) was achieved by the reaction of washed cells of Acetobacter aceti IFO 3281. The produced L-ribulose was then used as a substrate for the production of L-ribose and L-arabinose. The isomerization of L-ribulose to L-ribose and L-arabinose was carried out using L-ribose isomerase (L-RI) of Acinetobacter sp, strain DL-28 and L-arabinose isomerase (L-AI) of Mycobacterium smegmatis, respectively. At equilibrium, the ratio of L-ribose : L-ribulose was 70 : 30 and that of L-arabinose : L-ribulose was 90 : 10. After a simple purification treatment, both pentoses could be crystallized without the use of column chromatography. The crystals were confirmed as L-ribose and L-arabinose by High-performance liquid chromatography (HPLC), Infrared (IR), Nuclear magnetic resonance (NMR) and optical rotation measurements.