This paper demonstrates the development of pH and thermo-responsive fluorescent nanoparticles, which are composed of graphene oxide (GO) with BODIPY conjugated PEG, to trigger the detection of cancer cells through imaging based on intracellular accommodation. Responsiveness to pH is studied using atomic force microscopy and apparent thickness differences are seen with changes in pH. Confocal images of the nanoparticles (NPs) exhibit remarkably bright fluorescence at lysosomal pH, while no fluorescence is observed under a physiological environment, making the NPs a novel fluorescent probe. The NPs are able to accumulate the hydrophobic anticancer drug DOX due to the hydrophobic surface of GO and show excellent drug release behavior. Therefore, the NPs developed are novel candidates for a fluorescent probe to identify cancer cells and a drug carrier for cancer therapy.