Concentrically arranged multilayered fullerenes exhibiting onion-like morphology are popularly known as carbon nano-onion (CNO) and are useful in bioimaging application. On the basis of the origin of the fluorescence, the CNO-based nanoprobes are classified into type I and type II. The type I CNO-based nanoprobe needs a secondary moiety such as organic dyes or an amine functionalization at its surface to induce the fluorescence. On the other hand, the emission in type II does not originate from such an external surface passivating agent. The CNO-based system not only shows structural similarity to the well-known multiwalled carbon nanotube but is also a bit more advantageous because of its low cytotoxicity. These features enable their prolonged use in the biological system for imaging purposes. In particular, we have covered the aspects of synthesis, surface functionalization, the origin of fluorescence, and biocompatibility. In addition, recent developments directed toward in vitro and in vivo imaging studies by utilizing CNO-based nanoprobes are summarized here.