Understanding the interactions of nanomaterials(NMs) with biomolecules, organelles, cells, and organic tissues at the nano-bio interface can offer important information for their uptake, distribution, translocation, metabolism and degradation in vitro and in vivo, which can help to precisely tune and design “smart” NMs for biomedical applications. However, probing the interactions at the nano-bio interface, which generally requires dedicated analytical methods and tools, is remarkably complicated due to the dynamically changed nature of the nano-bio interface. Because of the advantages of high spatial resolution, high sensitivity, excellent accuracy, low matrix effects and non-destructiveness, synchrotron radiation(SR)-based analytical techniques have become extremely valuable tools. Herein, we present a comprehensive overview of SR-based techniques for the visualized study of NMs at cellular and subcellular interfaces and their transformation in vitro; the exploration of biodistribution, translocation, metabolism and degradation of NMs in vivo; and clarification of the molecular mechanisms of NMs’ reactions with biomolecules. Rapid development of advanced light source means that in situ, real-time analysis of NMs at the nano-bio interface will be achieved. Understanding the interactions of nanomaterials (NMs) with biomolecules, organelles, cells, and organic tissues at the nano-bio interface can offer important information for their uptake, distribution, translocation, metabolism and degradation in vitro and in vivo, which can help to precisely However, probing the interactions at the nano-bio interface, which typically requires dedicated analytical methods and tools, is remarkably complicated due to the dynamically changed nature of the nano-bio interface. Because of the advantages of high spatial resolution, high sensitivity, excellent accuracy, low matrix effects and non-destructiveness, synchrotron radiation (SR) -based analytical techniques have become extremely valuable tools. Herein, we present a comprehensive overview of SR-based techniques for the visualized study of NMs at cellular and subcellular interfaces and their transformation in vitro; the exploration of biodistribu tion, translocation, metabolism and degradation of NMs in vivo; and clarification of the molecular mechanisms of NMs’ reactions with biomolecules. Rapid development of advanced light source means that in situ, real-time analysis of NMs at the nano-bio interface will be achieved