Laccases are versatile enzymes employed in the degradation of pollutants and in organic synthesis reactions. Their immobilization enables the reuse of these biocatalysts, thereby reducing the overall process costs. However, studies on the immobilization of laccases onto polymers containing diazonium groups remain limited in the literature. This work investigates the relationship between experimental parameters—namely pH, ionic strength, and contact time between the enzyme solution and diazonium-functionalized polystyrene—and their effects on immobilization yield and biocatalyst activity in the oxidation of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS). A five-level experimental design was employed, varying the ionic strength of the citrate–phosphate buffer, pH, and contact time. The correlation between reaction variables and response variables was best fitted using second-order models. The variables pH and its quadratic interaction significantly affected the immobilization yield, while ionic strength, pH, and their interaction were significant in determining the activity of the resulting biocatalysts. The validity of the proposed regression models was confirmed by adjusted R2 values, F significance, and residual analysis. After two, three, four, five, and six reuse cycles, the catalytic activity losses were 33%, 46%, 50%, 59%, and 51% of the initial activity, respectively. The loss of catalytic activity between reuse cycles was low, suggesting that the material is suitable for extended reuse. Furthermore, after 120 days of storage, the biocatalyst retained 89.9% of its initial relative activity, indicating good stability when stored under refrigeration.
