Luminescent organic dots (O-dots) were synthesized via a one-pot, solvent-free thermolysis of citric acid in urea melt. The influence of the ratio of the precursors and the duration of the process on the properties of the O-dots was established and a mechanism of their formation was hypothesized. The multicolour luminescence tunability and toxicity of synthesized O-dots were extensively studied. The possible applications of O-dots for alive/fixed cell staining and labelling of layer-by-layer polyelectrolyte microcapsules were evaluated.