Abstract: in order to explore the mechanism of tobacco response to drought stress and to provide a basis for drought resistance screening in tobacco, an experiment was carried out to study the changes of MDA and proline contents and protective enzyme activities in leaves of flue-cured tobacco varieties Zhongyan 100 and Zhongyan under different PEG-6000 concentrations. The results showed that, MDA showed on a trend of increase with the time of stress treatment and at the same treating time, MDA content was the highest under the 15% concentration of PEG. Proline increased with the time of treatment and at the same treating time, proline content was the highest under the 15% concentration of PEG. The activities of superoxide dismutase (SOD), peroxdase (POD) and catalase (CAT) increased firstly and decreased subsequently with stress time, reaching the maximum activities after 1 day, with the highest activities under the 15% concentration of PEG. Zhongyan 14 was confirmed to have higher drought resistance, suggesting that the detection indexes in this study can be used in drought resistance evaluation of tobacco.