Characterization of materials at the nanoscale plays a crucial role in in-depth understanding the nature and processes of the substances. Mass spectrometry (MS) has characterization capabilities for nanomaterials (NMs) and nanostructures by offering reliable multi-dimensional information consisting of accurate mass, isotopic, and molecular structural information. In the last decade, MS has emerged as a powerful nano-characterization technique. This review comprehensively summarizes the capabilities of MS in various aspects of nano-characterization that greatly enrich the toolbox of nano research. Compared with other characterization techniques, MS has unique capabilities for real-time monitoring and tracking reaction intermediates and by-products. Moreover, MS has shown application potential in some novel aspects, such as MS imaging of the biodistribution and fate of NMs in animals and humans, stable isotopic tracing of NMs, and risk assessment of NMs, which deserve update and integration into the current knowledge framework of nano-characterization.