The biocatalytic system laccase/TEMPO/O2has attracted the attention of researchers over the past two decades. A variety of applications for the system include organic synthesis, modification of cellulose, and oxidative degradation of environmental contaminants. A rigorous and predictable quantitative assessment of the change in enzymatic activity under the influence of a mediator is important for such a system. In this study, the operative conditions for carrying out a model reaction for the degradation of the synthetic dye Indigo Carmine in the presence ofTrametes versicolorlaccase/TEMPO were determined and the enzyme inactivation under the action of a mediator and substrate was studied. The long-term stability ofTrametes versicolorlaccase was assessed and the regression model of the response surface of laccase activity under the influence of TEMPO was created. It has been shown that laccase is inactivated in the presence of TEMPO, but the addition of the dye, CuSO4or CuCl2reduces this effect. The system under study can be used repeatedly for the Indigo Carmine decolorization, however, a gradual falling rate during the process is observed from cycle to cycle. This is due to two reasons – firstly, a decrease in the enzyme activity with each batch and secondly, the consumption of the mediator (22% within 5 days). Relatively high enzyme activity (>40%) is maintained after 73 cycles (1 portion of IC contained 25 μM) using 500 μM TEMPO and 0.12 U mL−1laccase. The laccase/TEMPO system has shown its effectiveness in the treatment of artificial wastewater containing high concentrations of Indigo carmine (0.5 g L−1). In this case, the dye solution becomes 100% colorless within 5 hours in the presence of dye bath components and within 7.5 hours in a buffer solution.