Display omitted •A dual-signal responsive sensor was designed to detect GSH based on MnO2 nanoflakes.•This sensor combined the advantages of RRS and colorimetry for the detection of GSH.•The sensor was successfully applied to detect GSH in the drug samples.•This work broadens the application of RRS technique in the analytical field. Glutathione (GSH) plays a critical role in the living system and its abnormalities are closely associated with numerous clinical diseases. Thus, monitoring and detecting the content of GSH in the living system is still of great importance. Herein, a novel dual-signal responsive optical sensor is developed by using resonance Rayleigh scattering (RRS) and colorimetry for sensitive detection of GSH. In this sensor, MnO2 nanoflakes are used as both GSH recognizer and signal transducer of RRS and colorimetry. The solution of MnO2 nanoflakes shows strong RRS and absorption signals because MnO2 nanoflakes possess a large surface area and high molar extinction coefficient. However, MnO2 can be reduced to Mn2+ and MnO2 nanoflakes can be etched to small nanoparticles by GSH, causing both the RRS and absorption signals to decrease. Based on the change of RRS signal and absorbance of MnO2 nanoflakes, a novel dual-signal responsive optical sensor is successfully constructed to detect the content of GSH. The as-developed optical sensor toward GSH presents a favorable sensitivity with a detection limit of 0.033 and 0.67 μM for RRS and colorimetry, respectively. Furthermore, the as-developed approach is straightforward, quickly responsive, free-label, and cost-effective. More significantly, this method combines the advantages of RRS and colorimetry for the detection of GSH. Beyond this, the proposed RRS method has also been successfully utilized to detect the content of GSH in glutathione injection samples.