Biodegradation of environmentally hazardous synthetic dyes by enzymes has been achieved the highest interest in recent years. In this work, we optimized Remazol Brilliant Blue R (RBBR) dye biodegradation by Arthrographis kalrae derived laccase via the Box-Behnken design (BBD) approach of the surface response methodology (RSM). Optimization of dye decolourisation by one variable at a time (OVAT) approach resulted in optimal dye decolourisation at laccase dose (2 IU mL−1), pH (7.0), temperature (35 °C), incubation time (240 min), and initial dye concentration (100 mg L-1). The optimized process through BBD enhanced dye decolourisation (97.18%). Fourier Transform Infrared Spectroscopy and UV–Visible Spectrophotometry have proven biodegradation. In addition, in comparison to untreated samples, the laccase-treated dye sample showed relatively less phyto- and cytotoxic effect on Allium cepa L. Extra Precision Glide docking exhibited the binding affinity score of −5.355 kcal mol−1, between laccase-RBBR complex.