Abstract: We have developed a flow injection system with colorimetric detection to measure 1,5-anhydro-D-glucitol in serum. Serum samples are directly and serially injected into a clean-up column every 3.5 min to remove interferences before the enzymatic reaction. 1,5-Anhydro-D-glucitol, after being passed through the column, is oxidized by immobilized pyranose oxidase (EC 1.1.3.10), and the hydrogen peroxide produced reacts with the chromogen substrate in the presence of immobilized horseradish peroxidase (EC 1.11.1.7) to form Bindshedler's Green. The detection limit was 1.2 µmol/L (1.2 pmol). The correlation between results obtained with the present system (y) and gas chromatography- mass spectrometry (GC-MS) (x) in samples containing < 30 µmol/L 1,5- anhydro-D-glucitol, including many samples from patients with diabetes mellitus, was y = 0.975x-0.111 µmol/L (r = 0.997), which was superior to that obtained between the enzymatic and GC-MS methods. Our system needs only to be set up; it runs without any manual pretreatment, assays 17 samples/h, and shows imprecision (CV) of < 2%.