Highly photoluminescent carbon dots have been prepared in a one step procedure by hydrothermal treatment of formaldehyde at 180 °C. They show green fluorescence under UV light exposure and emission spectra are centered at 440 nm. Fluorescence lifetimes comprise between 0.7 and 2.70 ns, when the synthesis process lasted for 1-7 days. TEM images of nanoparticles showed a homogeneous size/shape distribution. When the thermal treatment process was carried out for a long time (30 days) formation of aggregates occurred. Carbon dots were further analyzed using (1)H and (13)C-NMR, Raman and FTIR spectroscopy techniques and XPS. Cell imaging of nanoparticles was carried out by using mouse MC3T3-E1 pre-osteoblasts as a model. The nanoparticles were selectively localized in the cytoplasm without further functionalization and could be realized by cellular phagocytosis, so that the fluorescence of these can be used for live cell imaging in vitro.