Alcohol oxidase catalyzes the oxidation of short lines alcohol to aldehyde. In this study, alcohol oxidase from Hansenula polymorpha (HpAOD) was induced by addition of 0.5% methanol as the carbon source and purified to electrophoretic homogeneity by column chromatographies. The purified HpAOD was immobilized with DEAE-cellulose particles and its biochemical properties were compared with those of free enzyme. The substrate specificity and the optimum pH of immobilized enzyme were similar to those of free enzyme. On the other hand, the Km values of free and immobilized enzymes for ethanol were 6.66 and 14.65 mM, respectively. The optimum temperature for free enzyme was ${50^{circ}C}$, whereas that for immobilized enzyme was ${65^{circ}C}$. Immobilized enzyme showed high stability against long storage. Immobilized enzyme was also tested for the enzymatic determination of ethanol by the colorimetric method. We detected 1 mg/liter ethanol ($1{ imes}10^{-4}$% ethanol) by 2,6- dichloroindophenol system. Therefore, the present study demonstrated that immobilized HpAOD has high substrate specificity toward ethanol and storage stability, which may be of considerable interest for alcohol biosensor and industrial application.