Investigations of the interactions between carbon nanomaterials (CNMs) and living organisms and their subsequent biological responses are of fundamental significance for toxicity assessment and further biomedical applications. In this work, the cellular uptake and cytotoxicity of multi-walled carbon nanotubes (MWCNTs), graphene oxide (GO) and nanodiamond (ND) were examined and compared. We demonstrated that all of the CNMs were readily internalized by HeLa cells through nonspecific cellular uptake. Their cell uptake ratios showed significant differences in the following order: ND > MWCNTs > GO. A series of biological assays were used to evaluate the cytotoxicity of CNMs. It was found that CNMs showed dose- and time-dependent cytotoxicity to HeLa cells. However, cytotoxicity of CNMs was not associated with their cell uptake ratios. Among them, ND exhibited the highest cell uptake ratio and the least cytotoxicity. To the best of our knowledge, this is the first study which has quantitatively determined and compared the cell uptake ratios and cytotoxicities of MWCNTs, GO and ND. And we expect that these results described here could provide useful information for the development of new strategies to design efficient drug delivery nanocarriers and therapeutic systems as well as deep insights into the mechanism of CNMs’ cytotoxicity.