AbstractStenotrophomonas maltophilia AAP56, laccase-producing bacteria, growing under different conditions, exhibit laccase activity that is highly affected by some environmental factors. Response surface methodology (RSM) was applied for the determination of laccase factor dependence using two substrates: ABTS (2,2’-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)) and DMP (2,6 Dimethoxy-phenol). RSM was carried out with a 24 factorial design using four variables, namely, dye (0 to 0.1 mg mL-1), Cu in Med (0 to 400 µM), shaking (0 to 150 rpm), and CuSO4 in assay (0 to 0.2 mM). Significant correlation between the effects of these variables on R1 (ABTS oxidase activity) and R2 (DMP oxidase activity) responses was detected. Astonishing results showed differences between these two activities with respect to copper activity dependence. Anoxic conditions exhibited a significant ability to induce the enzyme. This bacterial laccase activity (produced under optimal conditions according to RSM) was used to decolorize an azoic dye, Reactive Black 5 (RB5). It was efficient only in the presence of a redox mediator to degrade RB5 after 20 min of incubation time.