The durability of enzymatic electrochemical devices depends on the durability of enzymes and on that of mediators that shuttle electrons between enzymes and electrodes. The degradation of 2,2’-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) in the presence of laccase was evaluated in this study. The evaluation of the ABTS-adsorbed electrode in the presence and absence of laccase showed that the redox peak area of ABTS decreased to approximately 40% of the initial value with laccase, whereas the retention of the peak area was approximately 90% without laccase. The characterization of the degradation products in a mixed solution of ABTS and laccase was then performed using Fourier transform (FT)-IR, 1H-NMR, 13C-NMR, and electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS). The comparison of the spectra of the products of the mixed solution of ABTS and laccase with those of the solution containing only ABTS or laccase showed the formation of nitrosamine from FT-IR spectra and newly appeared sp2 and sp3 carbon from 13C-NMR spectra. By also considering the peaks observed in ESI-TOF-MS spectra, a possible degradation mechanism was proposed to be initiated by the adduct formation of the ABTS radical with water.