Glutathione (GSH) serves vital cellular biological functions, and its abnormal levels are associated with many diseases. To better understand its physiological and pathological functions, efficient methods for monitoring of GSH in living systems are desired. Although quite a few small molecule-based and nanomaterial-based one photon fluorescence probes have been reported for GSH, two-photon (TP) probes, especially nanoprobes with good membrane permeability, are more favorable for bioimaging applications, since TP fluorescence imaging can provide improved spatial localization and increased imaging depth. In this work, we for the first time reported a "turn-on" TP fluorescence nanoprobe for efficient detection of GSH in aqueous solutions and TP excited fluorescence imaging of GSH in living cells and tissues. The nanoprobe consists of two-photon mesoporous silica nanoparticles (TP-MSNs) with a large TP excitation action cross-section (Φδ) value of 103 GM and MnO2 nanosheets, which show intense and broad optical absorption and could act as efficient quenchers for TP fluorescence. In the sensing system, the negatively charged MnO2 nanosheets are adsorbed on the positively charged MSNs through electrostatic interaction, resulting in efficient quenching of their fluorescence, with very low background fluorescence observed. The addition of GSH could reduce MnO2 into Mn(2+), lead to the decomposition of the MnO2 nanosheets, and thereby result in remarkable enhancement of both one photon and TP excited fluorescence of the nanosystem. The nanoprobe shows a highly sensitive response to GSH in aqueous solutions, with a detection limit of 200 nM achieved. It also exhibits a high selectivity toward GSH relative to other biomolecules and electrolytes, with good membrane permeability and excellent biocompatibility. The nanoprobe was successfully applied in monitoring the change of the intracellular GSH in living cells and tissues via TP fluorescence imaging, demonstrating its value of practical application in biological systems.