The objectives of this study are to determine the function of the cotton ERFB3 subgroup transcription factor gene GhB301 after ectopic expression of tobacco (in resistance to Fusarium wilt). The overexpression of the GhB301 transgenic tobacco and wildtype tobacco treated with Fusarium wilt spore suspension, were used for analysis of the relationship between the defense enzyme activities and the expression of defenserelated genes and disease resistance before and after infection. The results showed that: (1) after treatment with Fusarium wilt for 15 days, the degree of yellowing of tobacco leaves in two transgenic lines was lighter than that in wild type; (2) Compared with noninoculated controls, the overexpression of the GhB301 transgenic tobacco and wildtype tobacco leaves showed a drastic increase of the enzyme activities of the oxide enzyme (POD), phenylalanine ammonia lyase (PAL) and polyphenol oxidase (PPO), and the peak of enzyme activities appeared earlier than that of wild type material. POD, PAL and PPO activities in the leaves of transgenic materials peaked at 3 d, while the POD and PAL activity in leaves of wildtype materials reached a peak at 5 d; (3) The expression level of reactive oxygenrelated genes, ethylene (ET)/jasmonic acid (JA) pathwayrelated genes, and pathogenesisrelated genes were affected in both the transgenic materials OE1 and OE2 after inoculation with Fusarium wilt. It indicated that the ectopic expression of the GhB301 in tobacco activated the expression of defenserelated genes and increased the activities of the defense enzymes, thus enhancing the resistance of tobacco to Fusarium wilt.