Abstract An FAD-dependent N 1, N 12-diacetylspermine oxidase (DASpmOX), which seems suitable for enzymatic determination of the tumor marker N 1, N 12-diacetylspermine (DASpm), was isolated from Debaryomyces hansenii T-42. DASpmOX exhibited the most excellent specificity toward DASpm among all polyamine oxidases found to date, and the specificity for DASpm could be raised by adjusting the pH of the buffer and adding TritonX-100. In potassium phosphate (pH 7.0) with 0.3% TritonX-100, this enzyme did not have any detectable activity toward free polyamines, and the reaction rate of N 1, N 8-diacetylspermidine, N 1-acetylspermine, N 1-acetylspermidine, and N 8-acetylspermidine was only 19%, 7.8%, 7.8%, and 1.0% of that of DASpm, respectively. The gene encoding DASpmOX was cloned and expressed in Escherichia coli. The apparent k cat and K m values of recombinant enzyme for DASpm were found to be 158 s − 1 and 3.1 × 10 − 4 M under the conditions described above, respectively.