A new nanosheet fluorescent chemosensor (GO–NH–COUR) based on graphite oxide (GO) covalently functionalized with a 4-methyl-7-aminocoumarin (NH 2 -COUR) has been prepared. The GO were prepared by a modified Hummers method. The products were characterized by TEM, XRD, and FT-IR. The photophysical properties of GO, NH 2 –COUR and GO–NH–COUR in aqueous and ethanol solution have been investigated by UV/vis spectrophotometry, steady-state and time-resolved fluorometry. They reflect a large effect of the NH 2 –COUR substituent on the fluorescence characteristics of GO. In aqueous solution, GO–NH–COUR probes undergo protonation–deprotonation in the acid to basic pH range, producing intensity increases with acid to near-neutral pH range. The advantages of the newly prepared nanoparticles are that they offer good dispersion in aqueous solution and optical properties. Confocal microscopy experiments showed that GO–NH–COUR can be transfected into the living cells and applied for fluorescence imaging.