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Herein, a novel bimodal ratiometric probe for sensitive and selective detection of biothiols (including glutathione (GSH), cysteine (Cys) and homocysteine (Hcys)) was constructed, which was based on the redox reaction between manganese dioxide nanoflakes (MnO 2 NFs) and rhodamine (RhB) and biothiols. When MnO 2 NFs was added into RhB solution, RhB was oxidized to a series of derivatives, accompanying with the colorimetric color changing from purple to light pink and fluorescence changing from red to green. In the presence of GSH, Cys or Hcys, they could reduce MnO 2 NFs to Mn 2+ , thereby preventing the following oxidization of RhB and the corresponding color and fluorescence changes. The absorption intensity ratio and fluorescence intensity ratio showed good linear relationships with the concentrations of biothiols. The colorimetric detection limits for GSH, Cys and Hcys were 0.057 μM, 0.140 μM and 0.087 μM, respectively. And the fluorescence detection limits were 0.177 μM, 0.282 μM and 0.161 μM. More importantly, this probe was successfully applied to monitor the concentration of GSH/Cys/Hcys in human serum samples, with satisfactory recovery. Thus, this MnO 2 NFs-RhB platform can potentially be a candidate for the detection of biothiols., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)