In this study, we present a rapid and simple method for the separation and direct detection of glutathione by combining gold nanoparticles and MALDI–TOF-MS with graphene as matrix. Gold nanoparticles enable the selective capture of thiol-containing compounds. Gold nanoparticles bound with analytes can be mixed with graphene matrix for direct analysis by MALDI–TOF-MS, which can avoid sample loss and contamination during transfer process. Compared with a conventional matrix, α-cyano-4-hydroxycinnamic acid, graphene exhibits an excellent desorption/ionization efficiency, thermal and mechanical properties. The use of graphene as matrix avoids the fragmentation of analytes. Stable analysis was achieved with less background interference even at the concentration of 0.625 ng/μL. To further confirm its efficiency, the optimized approach was applied to the separation and detection of glutathione in mouse liver extraction. This result showed the great potential of detection of biologically important thiols in biochemical and biomedical research.