Nitrobenzene (NB) is a synthetic hydrocarbon widely used in industries which is a highly toxic environmental pollutant. To investigate its genetic toxicity to plants, tobacco seedlings were exposed to varying concentration of NB solution for short-term (24 hours). The comet assay was used to examine the DNA damage in tobacco leaf cells under the stress of NB. The DNA migration increased with the dose of NB (from 10 to 100 mg/L). The values of tail intensity (T-DNA), tail moment (TM), and olive tail moment (OTM) increased linearly with NB concentrations between 5 mg/L and 100 mg/L. The results suggest that NB has genotoxic effect on the molecular level in tobacco leaf cells. The comet parameters were significantly different from the control when NB concentrations were higher than 5 mg/L ($P$ <0.05), suggesting that tobacco leaf cells have high sensitivity to NB stress. The phenomena demonstrated that the tobacco comet assay could be used efficiently monitor aquatic pollution caused by the toxic chemicals. In addition, a similar evaluation was performed using different NB and antioxidant ascorbic acid (Vc) mixed solutions. The DNA damage in the antioxidant group were significantly lower than those in the group without antioxidant, which illustrates that antioxidants could partially decrease the DNA damage induced by NB. These results suggest that oxidation reactions caused by NB might be one of the factors that lead to DNA damage in plants. However, to process the possible participation of mechanisms of DNA damage caused by NB, further investigation will need.