AimsThe increasing use of dyes leads to serious environmental concerns, it is significant to explore eco-friendly and economic approaches for dye decolorization. This study aimed to develop mediator cocktail (AS and ABTS) for enhancing the capability of laccase-mediator system in the removal of dyes. Methods and ResultsBy mediator screening, the mediators of ABTS and AS (ABTS, 2, 2-azino-bis-(3-ethylbenzothiazo-thiazoline-6-sulphonic acid); AS, acetosyringone) were combined for dyes decolorization. The Box-Behnken Design and response surface analysis was performed to optimize experiment conditions. Comparing the CotA-ABTS-AS cocktail system with CotA-single mediator system showed that the coupling of ABTS and AS could increase the decolorization rate 15 times higher, save a third of the cost and shorten the reaction time by 50%. In addition, our studies revealed that sequential oxidation may occur in CotA-ABTS-AS system. ConclusionsCompared with CotA laccase-single mediator system, the CotA-ABTS-AS cocktail system showed advantages including higher efficiency, lower cost and shorter reaction time. Significance and Impact of the StudyThis was the first report on the dyes decolorization by laccase mediator cocktail system. These results paved the curb for the application of laccase mediator system in various industrial processes.