Graphene is an increasingly important nanomaterial that has shown great promise in the area of nanotechnology. In this study, fluorescein-functionalized graphene oxide (GO) is synthesized via a polyethylene glycol (PEG) bridge and its application in intracellular imaging is explored. GO is an oxide form of graphene that provides an ideal platform to prepare graphene-based functional nanomaterials via chemical modification. The PEG bridge was introduced to prevent GO-induced quenching of conjugated fluorescein. The fluorescein–PEG–GO conjugate thus prepared exhibits excellent pH-tunable fluorescent properties and, more significantly, can be efficiently taken up by cells and serve as a fluorescent nanoprobe for intracellular imaging.