Background: Azo dyes are the most widely used synthetic colorants in the textile, food, pharmaceutical, cosmetic, and other industries, accounting for nearly 70% of all dyestuffs consumed. Recently, much research attention has been paid to efficient monitoring of these hazardous chemicals and their related metabolites because of their potentially harmful effect on environmental issues. In contrast to the complex and expensive instrumental procedures, the detection system based on the QDs with the superior optochemical properties provides a new era in the pollution sensing and prevention. Methods: We have developed a QD-enzyme hybrid system to probe MR in aqueous solutions using a fluorescence quenching procedure. Results: The azoreductase enzyme catalyzed the reduction of azo group in MR, which can efficiently decrease the FRET between the QDs and MR molecules. The correlation between the QDs photoluminescence recovery and MR enzymatic decolorization at the neutral phosphate buffer permitted the creation of a fluorescence quenching-based sensor. The synthesized biosensor can be used for the accurate detection of MR in a linear calibration over MR concentrations of 5-84 μM, with the LOD of 0.5 μM in response time of three minutes. Conclusion: Our findings revealed that this fluorometric sensor has the potential to be successfully applied for monitoring a wide linear range of MR concentration with the relative standard deviation of 4% rather than the other method.