Glutathione (GSH) is an important antioxidant biomarker with pivotal roles in multiple biological processes. Herein, a novel dual-emission ratiometric fluorescence nanoprobe was developed for selective and sensitive GSH detection by exploiting competitive interactions with a silver-riboflavin (Ag-RF) complex. The nanoprobe consists simply of a mixed solution of Ag-RF complex displaying characteristic yellow emission at 525 nm and nitrogen-doped carbon dots exhibiting blue emission at 440 nm. Due to the strong affinity of GSH toward silver ions, the addition of GSH liberates riboflavin from the Ag-RF complex leading to quenching of adjacent nitrogen-doped carbon dots through fluorescence resonance energy transfer (FRET). By ratiometrically measuring the fluorescence intensities at 525 and 440 nm (F₅₂₅/F₄₄₀), GSH levels can be rapidly quantified without interference. This sensor gave excellent linearity (R² = 0.9986) over 0.05–70 μmol/L GSH with high sensitivity (limit of detection = 0.015 μmol/L). The sensor also provided accurate GSH analysis in spiked human serum samples (96–98.5 % recoveries), validating practical applicability. With advantages of simplicity, selectivity, and low sample volume requirements, this fluorescent nanoprobe holds great promise as a tool for real-time, in-field monitoring of GSH for basic research and clinical investigations.