Photoluminescent graphene quantum dots (GQDs) have fascinating optical and electronic properties with numerous promising applications in biomedical engineering. In this work, we first studied the in vivo biodistribution and the potential toxicity of carboxylated photoluminescent GQDs. KB, MDA-MB231, A549 cancer cells, and MDCK normal cell line were chosen as in vitro cell culture models to examine the possible adverse effects of the carboxylated photoluminescent GQDs. The carboxylated GQDs are desirable for increased aqueous solubility. All cancer cells efficiently took up the carboxylated GQDs. No acute toxicity or morphological changes were noted in either system at the tested exposure levels. A long-term in vivo study revealed that the GQDs mainly accumulated in liver, spleen, lung, kidney, and tumor sites after intravenous injection. To reveal any potential toxic effect of the GQDs on treated mice, serum biochemical analysis and histological evaluation were performed. The toxicity results from serum biochemistry and complete blood count study revealed that the GQDs do not cause appreciable toxicity to the treated animals. Finally, we observed no obvious organ damage or lesions for the GQDs treated mice after 21 days of administration at 5 mg/kg or 10 mg/kg dosages. With adequate studies of toxicity, both in vitro and in vivo, photoluminescent GQDs may be considered for biological application.