Abstract: To explore the preparation method of carbon dots from tobacco stalks and evaluate their anti-tobacco mosaic virus (TMV) activity, four types of biomass carbon dots were prepared via a one-step solvothermal method using tobacco stalks as carbon source and acetone, ultrapure water, ethanol and methanol as solvents, respectively. The microstructures, crystal structures, surface functional groups and electric charges of the carbon dots were characterized using transmission electron microscopy (TEM), X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FT-IR) and Zeta electric potential. Their optical properties were examined using ultraviolet-visible and fluorescence spectrophotometers, and their anti-TMV activity was evaluated using the detached leaf disc method. The results showed that: 1) All four types of carbon dots exhibited a sphere-like structure with homogeneous dispersion and without significant agglomeration. Their surfaces were rich in hydrophilic functional groups such as carboxyl and hydroxyl groups and carried negative charges. Those structural features endowed carbon dots with good water solubility and stable optical properties, enabling them to emit sustained fluorescence under ultraviolet excitation. 2) All carbon dot samples exhibited inhibitory effects against TMV, and their antiviral effects increased with increasing concentration. The carbon dot prepared with ethanol exhibited better antiviral activity.