In this study, we investigated the graphene oxide (GO) nanosheets of specific interaction with metabolome in breast cancer cells. The edge-oxidized form of GO nanosheets have multiple applications in biomedical research. At present, the actual metabolic mechanisms of GO nanosheets is not yet clearly understood. Here, we describe the effects of GO exposure on MCF-7 breast cancer cells using 1H nuclear magnetic resonance (1H-NMR) profiling. We treated MCF-7 cells with different GO concentrations and examined the metabolic distributions. Interestingly, we found that the treatment affected arginine metabolism, proline metabolism, and aminoacyltRNA biosynthesis, including anabolism and catabolism. GO dose-dependently increased the number of metabolic disturbances in cancer steroids. Therefore, these data suggest that GO can significantly potentiate the metabolic effect in MCF-7 cells. These results demonstrate that using targeted metabolomics profiling and pattern recognition analysis display the potential metabolic changes and underlying metabolic impacts in human breast cancer cells.