Manufactured nanomaterials with novel physicochemical properties are an important basis for nanosciences and related technologies. Nanotoxicology, aiming to understand the principles of interactions at the nano-bio interface and the relationship between the physicochemical properties of nanomaterials and their toxicological profiles, has become a new frontier in nanoscience. Nearly one decade of nanotoxicology research has shown that the interactions between nanomaterials and proteins, cells, animals, humans and the environment as well as the underlying mechanisms of toxicity for nanomaterials are remarkably complicated, requiring dedicated analytical methodology and tools. Because of their advantages of absolute quantification, high sensitivity, excellent accuracy and precision, low matrix effects and non-destructiveness, nuclear analytical techniques have been playing important roles in the study of nanotoxicology. A systematic summary and comprehensive review of the advanced nuclear analytical and related techniques in nanotoxicology is greatly needed. In this review article, we present a comprehensive overview of nuclear analytical techniques applied to the physicochemical characterization of nanomaterials, structural analysis of bio-nano interactions, visualization of nanomaterials in vitro, quantification of bio-distribution, bio-accumulation, and transformation of nanomaterials in vivo. As important complementary tools, optical imaging technologies are also highlighted. Future directions regarding advanced nuclear analytical approaches for nanotoxicology are also discussed. The rapid development of advanced light source-based techniques will enable new high-throughput screening techniques and provide high sensitivity with low detection limits, which are required for the distribution, imaging, and structural analysis of nanomaterials, and the molecular information of biomarkers for all aspects of nanotoxicology.