The effect of various physicochemical parameters was investigated on the removal efficiency of azo dye reactive red 120 from wastewaters by sunflower hairy roots. The highest percentage removal of the colored dye from wastewater(s) was observed at 25 °C, pH 7, adsorbent dosage 12.5 g L−1, shaking speed 85 rpm and light intensity 20 μmol m−2 s−1. Further, a combinatorial study with all the optimized parameters was performed in the dye concentration range of 20–110 mg L−1 to determine the synergistic effect, if any, of all the physicochemical parameters on the kinetics of the dye removal. Nearly 100% dye removal could be achieved in 32 h as opposed to only 54% under the unoptimized conditions. Similarly, the initial dye removal rate could also be enhanced (from 3.4 to 19.6 mg L−1 h−1) under the optimized conditions. This correlated well with the proportionate increase in the (enzymatic and non-enzymatic) antioxidant potential of the root system under the optimized process parameters. The potential degradation of reactive red 120 by sunflower hairy roots was studied by Fourier transformed infrared spectroscopy and scanning electronic microscope analysis. Interestingly, sunflower hairy root system was also able to remove other azo dyes like reactive orange 16 and reactive blue 21 from their aqueous solutions. As seen in the study with reactive red 120, the removal increased from 49.5% under unoptimized conditions to 80.7% under optimized conditions (1.6 fold) for reactive orange 16 and from 55 to 82% (1.5 fold) for reactive blue 21, suggesting that these physicochemical parameters could significantly influence the removal of azo dyes in plant cells and their optimization can lead to further enhancement in the efficiency of the phytoremediation process for azo dye removal. The effect of optimized parameters on the removal of azo dyes from other wastewater compositions (synthetic and actual with different total dissolved solids) was also investigated to check the extrapolation of the key results obtained earlier with an aqueous solution of the dyes. As observed earlier, in a similar combinatorial study, the removal of reactive red 120 (90 mg L−1) could be improved from 72 to 92% (1.2 fold) in its solution in tap water (TDS: 650 ppm), from 26 to 72% (2.8 fold) in its solution in synthetic wastewater (TDS: 950 ppm) and from 41 to 62% in its solution in a secondary treated actual textile wastewater (TDS: 350 ppm). © 2021, Islamic Azad University (IAU).