Introduction: Carbon Dots (CDs) have emerged as promising nanomaterials for pharmaceutical and diagnostic applications due to their tunable fluorescence, biocompatibility, and potential for surface functionalization. Their dual applicability as drug delivery nanocarriers and fluorescent probes for bioimaging makes their physicochemical characterization crucial for advancing biomedical research. Methods: CDs were synthesized using citric acid as the carbon precursor through two distinct approaches: thermal pyrolysis in an oil bath at 200 °C for 30 min and a hydrothermal method at 200 °C for 4 h in the presence of urea as a nitrogen source. The nanostructures were characterized by transmission electron microscopy (TEM), UV-Vis absorption spectroscopy, photoluminescence spectroscopy, Fourier-transform infrared spectroscopy (FTIR), Raman spectroscopy, and zeta potential analysis. Results: TEM analysis revealed spherical and homogeneous particles with an average diameter of 3.0–3.5 nm, consistent with the size range of quantum dots. Optical characterization showed stable photoluminescence, with absorption maxima at 333–350 nm and excitation-independent emission attributed to ordered sp² domains. Nitrogen doping significantly enhanced fluorescence intensity, increasing quantum yield and improving stability within a physiological pH range (4.5–9.5). FTIR spectra confirmed the presence of surface functional groups such as C=O, C–N, and C–OH, while Raman analysis revealed the characteristic D and G bands of graphitic carbon. Zeta potential values of approximately –30 mV indicated high colloidal stability and abundant anionic groups favorable for further functionalization. Conclusions: The combination of small size (<10 nm), high photostability, tunable fluorescence, and negative surface charge highlights the multifunctional potential of CDs. These features support their application both as drug delivery systems, responsive to physiological conditions, and as fluorescent probes for bioimaging, reinforcing their relevance as advanced platforms in therapeutic and diagnostic contexts.