During the past decades, carbon dots (CDs) as a kind of nanoparticles with interesting fluorescence properties have retained their place as one of the best bioimaging agents, although their effects on plants have been rarely studied. In this study, we synthesized two kinds of concentration‐dependent multicolour CDs using two solvent approaches, phosphate‐buffered saline (PBS) and ethanol 20%. We confirmed the nature of the CDs through Fourier transform infrared spectroscopy, atomic force microscopy, dynamic light scattering, zeta potential, X‐ray powder diffraction, and high‐resolution transmission electron microscopy. Afterwards, the cytotoxicity, phytotoxicity, and bioimaging of animal cells and plants using both synthesized CDs were examined. Eventually, PBS‐based CDs were recommended during this study as an efficient bioimaging agent for animal cells and plants because of the appealing features of this CD, such as a small size range of less than 10 nm, surface charge with an average of −24 mV, a high quantum yield of 35.82%, the higher fluorescence intensity of ~400 a.u. for blue fluorescence light and 250 a.u. for green fluorescence light. Other features showing the superiority of PBS‐based CDs include high photostability, low phytotoxicity (p ≤ 0.05 and p ≤ 0.01) and above all, there was no significant cytotoxicity at the concentration range of 500–7.81 μg/ml.