This study investigates the biocompatibility of graphene oxide (GO) by examining its effects on human fibroblast cells and mice. GO was synthesized using the modified Hummers method and characterized by high-resolution transmission electron microscopy (TEM) and atomic force microscopy (AFM). Human fibroblast cells were cultured with different doses of GO for up to 5 days, and the cytotoxicity was assessed using CCK8 assays and cell adhesion assays. The results showed that GO doses below 20 μg/mL were non-toxic, while doses above 50 μg/mL exhibited significant cytotoxicity, including decreased cell adhesion, induced cell apoptosis, and accumulation in lysosomes, mitochondria, endoplasm, and the cell nucleus. In vivo experiments involved injecting GO into mice at low, medium, and high doses (0.1, 0.25, and 0.4 mg, respectively) via the tail vein. The mice were monitored for 1 day, 7 days, and 30 days. The low and medium doses (0.1 and 0.25 mg) did not show significant toxicity, while the high dose (0.4 mg) led to chronic toxicity, including 4 out of 9 mice dying and the formation of lung granulomas. Histopathological analysis revealed that GO accumulated primarily in the lungs, liver, spleen, and kidneys, with minimal accumulation in the brain and kidney. The study concludes that GO exhibits dose-dependent toxicity to both cells and animals, with potential mechanisms involving cell signaling, energy metabolism, and gene transcription. The findings highlight the need to consider the biocompatibility of GO in biomedical applications, particularly in vivo, due to its long-term accumulation and difficulty in clearance by the kidney.This study investigates the biocompatibility of graphene oxide (GO) by examining its effects on human fibroblast cells and mice. GO was synthesized using the modified Hummers method and characterized by high-resolution transmission electron microscopy (TEM) and atomic force microscopy (AFM). Human fibroblast cells were cultured with different doses of GO for up to 5 days, and the cytotoxicity was assessed using CCK8 assays and cell adhesion assays. The results showed that GO doses below 20 μg/mL were non-toxic, while doses above 50 μg/mL exhibited significant cytotoxicity, including decreased cell adhesion, induced cell apoptosis, and accumulation in lysosomes, mitochondria, endoplasm, and the cell nucleus. In vivo experiments involved injecting GO into mice at low, medium, and high doses (0.1, 0.25, and 0.4 mg, respectively) via the tail vein. The mice were monitored for 1 day, 7 days, and 30 days. The low and medium doses (0.1 and 0.25 mg) did not show significant toxicity, while the high dose (0.4 mg) led to chronic toxicity, including 4 out of 9 mice dying and the formation of lung granulomas. Histopathological analysis revealed that GO accumulated primarily in the lungs, liver, spleen, and kidneys, with minimal accumulation in the brain and kidney. The study concludes that GO exhibits dose-dependent toxicity to both cells and animals, with potential mechanisms involving cell signaling, energy metabolism, and gene transcription. The findings highlight the need to consider the biocompatibility of GO in biomedical applications, particularly in vivo, due to its long-term accumulation and difficulty in clearance by the kidney.