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Fluorescent carbon dots (CDs) with advantages including excellent biocompatibility, low cost, and easy modification make them highly promising for applications in biological imaging. Here, we successfully synthesized dark green fluorescent CDs (g‐CDs) using trinitropyrene as a carbon source and 2,4‐diaminobenzenesulfonic acid as the reactant in one‐pot synthesis. The results revealed that the g‐CDs had an average uniform particle size of 4.93 nm, with a structure similar to graphite. The g‐CDs imparted excellent water‐solubility due to the presence of hydrophilic groups. The g‐CDs exhibited an optimal fluorescence emission wavelength of 512 nm in acetone solvent, with a maximum quantum yield of 38.4%. When transferred into a normal saline solution, the g‐CDs retained their green fluorescence, with the optimal fluorescence emission wavelength shifting to 505 nm. Notably, we observed a significant increase in fluorescence intensity and maximum quantum yield of up to 79.4% when the g‐CDs were dissolved in a polar solvent. Furthermore, in vitro fluorescence imaging experiments demonstrated rapid cell entry, excellent fluorescence performance, high biocompatibility, and universality of the g‐CDs. These findings underscore the high hydrophobicity and biocompatibility of our newly designed g‐CDs, thus highlighting their immense potential as fluorescent probes in bioimaging and disease tracing applications.