The authors describe a sensitive method for determination of glutathione (GSH) that is based on a thiol-triggered inner filter effect on the fluorescence of N-doped carbon dots (N-doped CDs). N-doped CDs with a quantum yield as high as 31% were prepared by a one-pot procedure, and 5,5′-dithiobis-(2-nitrobenzoic acid) was employed as a reagent for GSH recognition. The reaction product (5-thio-2-nitrobenzoic acid; TNB) acts as an absorber of the 410-nm light used to photo-excite the N-doped CDs. Hence, the fluorescence of N-doped CDs (peaking at 510 nm) is reduced with increasing concentrations of GSH. As little as 30 nM of GSH can be detected by this method. The approach was successfully applied to (a) food analysis, (b) an investigation of an oxidative stress model, and (c) to live cells imaging. The method does not require the surface of N-doped CDs to be chemically modified, and a linkage between receptor and fluorophore is not needed. In our perception, the method may become a viable tool for the detection and imaging of thiols.