Given the research and applications of carbon nanomaterials (CNMs) in various fields, the safety of CNMs has attracted significant attention. As with any pollutant, the separation and analysis of CNMs in complex environmental and biological matrices are the prerequisites for the precise evaluation of nanosafety. However, compared with conventional pollutants and other nanomaterials, the methodology of separation and analysis of CNMs in complex matrix remains immature. To clarify the targeted CNMs for analysis, this review explored the transformation and degradation of CNMs in natural environments and organisms. The sample treatments, for example, extraction, separation and enrichment of CNMs, are then discussed. Subsequently, the available analytical approaches including microscopic, scattering, diffraction, spectroscopic and other techniques are illustrated and compared. Herein, the combined employment of diverse techniques is highlighted to minimize the individual limitations. Finally, the challenges and perspectives for the quantitative/qualitative determination of CNMs in environmental and biological matrices are proposed. (C) 2016 Elsevier B.V. All rights reserved.