A rapid enzymatic fluorometric assay for measuring d-arabinitol in serum was developed using recombinant d-arabinitol dehydrogenase from Candida albicans (rArDH). rArDH was produced in Escherichia coli and purified by dye-ligand affinity chromatography. rArDH was highly specific for d-arabinitol, cross-reacting only with xylitol (4.9%) among all polyols tested. A Cobas Fara II centrifugal autoanalyzer (Roche) was used to measure NADH fluorometrically when rArDH and NAD were added to serum extracts, and d-arabinitol concentrations were calculated from standard curves derived from pooled human serum containing known amounts of d-arabinitol. The method was precise (mean intra-assay coefficients of variation [CVs], 0.8%, and mean interassay CVs, 1.6%) and rapid (3.5 min per assay) and showed excellent recovery of added d-arabinitol in serum (mean recovery rate, 101%). The mean and median d-arabinitol/creatinine ratios were 2.74 and 2.23 μM/mg/dl, respectively, for the 11 patients with candidemia compared to 1.14 and 1.23 μM/mg/dl, respectively, for 10 healthy controls (P < 0.01). These results confirm earlier studies showing that serum d-arabinitol measurement may help to promptly diagnose invasive candidiasis. The technique shows a significant improvement in terms of accuracy, cost, simplicity, specificity, and speed compared with gas chromatography, mass spectrometry, and earlier enzymatic assays.