Reactive Blue 222 (RB222) is widely used in textile industries and hence a common recalcitrant pollutant in the industrial effluent. Bioremediation of this dye is of significance as its one of the complex dyes with high molecular weight. In the present study, we isolated a novel bacterial strain Enterobacter CU2004 from the industrial waste and characterize using16S rRNA gene sequencing. Its potential to dye degradation was evaluated in a simple minimal salt media with the parameters namely dye concentration (100-1000 ppm), pH (4-9), temperature (15-55°C), Carbon source (Lactose, Sucrose, Glucose, Starch, and Fructose), and Nitrogen source (Casein, Yeast extract, Peptone, Tryptone, Ammonium sulphate, and Urea) in a 24 h culture. Finally, data obtained were extended to design of experiment based optimization for the degradation efficacy of Enterobacter CU2004 and to validated design space was established. The novelty is in optimizing the design space parameters for highest percentage of degradation ≥90% by the bacterial isolate Enterobacter CU2004 were finalized as 30-37°C temperature, 133-249 ppm dye concentration, Lactose as Carbon source, Yeast extract as Nitrogen source, and the pH as 8. Microbial dye degradation was confirmed by FTIR, HPLC and GCMS studies. Further studies revealed the dye intermediates and the potential of Enterobacter CU2004 toward the degradation of complex, high molecular weight industrial dye RB222.