Recently, bacterial laccases has drawn researchers' interest due to their ability to overcome high pH and salt concentration conditions compared to fungal laccases. Here we report a novel micro-aerobic cultivation strategy for enhancing CotA laccase expression and study its application for dye decolorization. Micro-aerobic cultivation of Escherichia coli BL21 (DE3) strain carrying pT7-FLAG-MAT-TAG-1-CotA had significantly enhanced CotA laccase activity up to 13903 U/L. The most unique findings of this investigation are that micro-aerobic cultivation strategy enhanced the reactive oxygen species production which consequently led to the over expression of CotA laccase gene. Malachite green, Crystal violet, Congo red and Bromophenol blue were efficiently decolorized by using purified CotA laccase without presence of any mediators at pH 6 and 9. These results provide a great platform for the dynamic production and application of bacterial laccase in industry.