Pentaethylenehexamine and d-penicillamine co-functionalized graphene quantum dots (PEHA-GQD-DPA) was made via one two-step thermal pyrolysis. The resulting PEHA-GQD-DPA is composed of the graphene sheets with an average size of 3.16 nm and the rich of functional groups. It gives an ultra strong fluorescence emission with the fluorescent quantum yield of 90.91% and sensitive and selective optical response towards Hg2+. The fluorescence intensity linearly decreases with the increase of Hg2+ in the range of 1.0 × 10-10-2 × 10-4 M with the detection limit of 4.6 × 10-11 M (S/N = 3). No species tested interfere with detection of Hg2+. The fluorescence quenched by Hg2+ can be well recovered by glutathione. The fluorescence intensity linearly increases with the increase of glutathione in the range of 5 × 10-8-2.5 × 10-6 M with the detection limit of 1.7 × 10-8 M (S/N = 3). The PEHA-GQD-DPA as a fluorescence probe has been successfully applied in determination of Hg2+ in natural water and glutathione in human serum and SW480 cell imaging.