【Objective】 In order to provide the biochemical and metabolic basis of tobacco breeding for disease resistance and resistance identification. 【Method】Resistant (Yueyan 97) and susceptible (Changbohuang) tobacco cultivars were inoculated with Ralstonia solanacearum by trunk injection. Defensive enzyme activities and metabolites were measured by using colorimetric method and GC-MS.【Result】The activities of phenylanine ammonia lyase (PAL), superoxide dismutase (SOD), peroxidase (POD) and polyphenol oxidase (PPO) in the resistant cultivar ‘Yueyan 97’ were higher than those in the susceptible cultivar ‘Changbohuang’ with the same inoculation treatment. The PAL activities of Yueyan 97 and Changbohuang were higher than that in control on the seventh and fifth days after inoculation, respectively. The PAL activities of both cultivars were lower compared to control at any other time. For both cultivars, SOD, POD and PPO activities increased compared to control in the earlier days, but were lower compared to control during the later time period. The types of POD and PPO isozymes in the resistant cultivar ‘Yueyan 97’ were higher than those in the susceptible cultivar ‘Changbohuang’. The width and color of isozyme bands of Yueyan 97 were enhanced at 3 d after inoculation, while those of Changbohuang were not enhanced. It suggested that the resistant cultivar could quickly respond to external stimuli and accelerate the formation of defense-related substances. Detection of metabolites showed that the relative contents of some substances in Yueyan 97 such as myo-inositol, nicotine, malic acid, and L-threonine were higher compared to control, but in Changbohuang were lower compared to control, suggesting that cultivar differences in resistance against R. solanacearum might be related to these substances.【Conclusion】 These four defensive enzyme activities and the contents of the above metabolites could be used as biochemical indexes for evaluating tobacco resistance against R. solanacearum.