Fluorescence biosensors that enable highly sensitive detection of glutathione (GSH) are in great demand for various biological investigations and early disease diagnoses. Here, we report a turn-on fluorescence nanoplatform based on fluorescent semiconducting polymer nanoparticle@MnO2 nanosheets (P-dot@MnO2) nanocomposites for rapid homogeneous determination of GSH. The near-infrared (NIR) fluorescent P-dots were prepared by doping NIR dyes into polymer matrix and then encompassed by MnO2, which resulted in a remarkable fluorescence quenching. Owing to the selective decomposition of MnO2 by GSH, the fluorescence recovery was achieved in the presence of GSH. On the basis of the target-induced turn-on fluorescence response, the developed nanoplatform can readily detect GSH with a high sensitivity up to 0.26 μM, as well as a superior specificity. Furthermore, it was successfully applied in monitoring the intracellular GSH in living cells, revealing its great potential in biomedical applications.