The fluorescent carbon dot (C-dot) is a new class of carbon nanomaterials. It has a discrete or quasispherical structure, typically measures less than 10 nm and contains sp(2)/sp(3) carbon, oxygen/nitrogen-based groups and surface-modified functional groups. Compared with semiconductor quantum dots (QDs), C-dots offer much lower toxicity and a better biocompatibility profile. Their other favorable features include easy and inexpensive synthesis and surface modification potential. C-dots can be morphologically classified into graphene-based quantum dots (GQDs) and amorphous carbon nanodots (ACNDs). Numerous methods have been developed to synthesize C-dots, and are mainly divided into 'top-down' and 'bottom-up' routes. In the top-down route, C-dots (mostly GQDs) is derived from the separation of large carbon precursors. The 'bottom-up' method primarily involves the dehydration, polymerization and carbonization of small molecules to form the GQDs and ACNDs through thermal/hydrothermal synthesis, microwave irradiation, and solution chemistry. Potential applications of C-dots have been explored in a number of cellular and in-vivo imaging approaches. However, some difficulties remain, including limited penetration depth and poorly controlled in-vivo pharmacokinetics, which depends on multiple factors such as the morphology, physiochemical properties, surface chemistry and formulation of C-dots. The exact mechanism of in-vivo biodistribution, cellular uptake and long-term toxicological effect of C-dots still need to be elucidated. An integrated multi-disciplinary approach involving chemists, pharmacologists, toxicologists, clinicians, and regulatory bodies at the early stage is essential to enable the clinical application of C-dots.