A membrane-bound xylose oxidizing PQQ-dependent dehydrogenase from Gluconobacter oxydans was purified with a simple large-scale applicable purification procedure. The activity recovery from membrane extract was 33% with 130-fold purification. Important characteristics with respect to the application of the dehydrogenase in biosensor technology were studied. The purified enzyme was most stable in the pH range 3.5–6.5. The pH optimum for xylose oxidation was in the range 7.5–8 for the solubilized enzyme. Optimal pH for the electrochemical detection of xylose oxidation was 6.5. Dimethyl and carboxylic acid derivatives of ferrocene were able to mediate electrons transferred in xylose oxidation from the enzyme immobilized on graphite electrode to the electrode. Hence the purified enzyme appeared to be suitable for biosensor applications.