Although graphene oxide (GO) nanosheets are widely used in different fields, the mechanism of their toxicity remains relatively unknown. NMR‐based metabolomics was used to study in vivo time and dose‐dependent toxicity of GO nanosheets in mice. Sixty serum samples from mice in four different time intervals including 24 and 72 h and 7 and 21 days after injection of 0‐, 1‐, and 10‐mg/kg b.w. were analyzed based on 1HNMR spectra of each sample and multivariate methods. In comparison with the control group, 12 changed metabolites were identified in GO nanosheet‐treated mice groups. These metabolites are involved in steroid hormone biosynthesis and steroid biosynthesis pathways. It was seen that the time factor is more important than the dose factor and the groups were separated in a time direction, completely. We found that GO nanosheets has toxicity and can affect steroidal hormones. However, this study shows that after 21 days, the treated groups regardless of their GO nanosheet dose are very close to the control group. This means that in one step exposure to GO nanosheets, their toxicity diminished after 21 days.