Determination of glutathione (GSH) is closely related to the clinical diagnosis of many diseases. Thus, a fluorescent and colorimetric dual-readout strategy for the sensitive determination of glutathione was proposed. The mesoporous silica nanoparticle–gold nanocluster (MSN–AuNC) nanocomposites with significantly enhanced emission and effectively improved photostability characteristics were used as fluorescent probes. Based on the inner filter effect (IFE), the fluorescence of MSN–AuNCs at 570 nm can be effectively quenched by oxidized 3,3′,5,5′-tetramethylbenzidine (oxTMB) with absorption in the wavelength ranges of 330–470 nm and 500–750 nm. However, the addition of GSH could cause the reduction of blue oxTMB to colorless TMB, resulting in the inhibition of IFE and the recovery of the fluorescence of MSN–AuNCs. Therefore, using oxTMB as both quencher and color indicator, a dual-readout oxTMB/MSN–AuNC sensing system for the sensitive determination of GSH was constructed. As signal amplification is caused by the fluorescence enhancement of MSN–AuNCs, the detection limits as low as 0.12 μM and 0.34 μM can be obtained for fluorescent and colorimetric assay, respectively. This method may not only offer a new idea for the sensitive and effective determination of GSH, but also broaden the applications of AuNCs in fluorescent and colorimetric dual-readout bioanalysis.