Abstract: To explore the preparation approach of carbon dots from tobacco stems and evaluate their antiviral activities against tobacco mosaic virus (TMV), four types of biomass carbon dots were prepared via a one-step solvothermal method with acetone, ultrapure water, ethanol, and methanol as solvents, respectively. The microscopic morphology, crystal structures, surface functional groups, and charges of the CDs were characterized using transmission electron microscopy (TEM), X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FT-IR), and Zeta potential analysis. Their optical properties were examined using ultraviolet-visible and fluorescence spectrophotometers, and their antiviral activities against TMV was evaluated using the isolated leaf disc assay. The results showed that: 1) All four types of carbon dots exhibited a spherical-like structure, and they were uniformly dispersed with no significant agglomeration, and possessed a surface rich in hydrophilic functional groups, such as carboxyl and hydroxyl, as well as a negative charge. These structural characteristics endowed good water solubilities and stable optical properties, enabling them to produce sustained fluorescence under ultraviolet excitation. 2) All carbon dots exhibited inhibitory effects against TMV, with their activities showing clear concentration dependence: as the carbon dots’ concentrations increased, the antiviral efficacies continued to enhance. Among these, the carbon dot prepared using ethanol as the solvent demonstrated superior antiviral activity.