Carbon nanodots (C-dots) are recently discovered fluorescent carbon nanoparticles with typical sizes of <10 nm. The C-dots have been reported to have excellent photophysical and chemical characteristics. In recent years, the advances in the development and improvement in C-dots synthesis, characterization, and applications are burgeoning. In this review, we introduce the most commonly used techniques for the characterization of C-dots. The characterization techniques for C-dots are briefly classified, described, and illustrated with applied examples. In addition, the analytical separation methods for C-dots (including electrophoresis, chromatography, density gradient centrifugation, differential centrifugation, solvent extraction, and dialysis) are included and discussed according to their analytical characteristics. The review concludes with an outlook towards the future developments in the characterization and the analytical separation of C-dots. The comprehensive overview of the characterization and the analytical separation techniques will safeguard people to use each technique more wisely.