Abstract: Terpenoids play an important role in plant growth, development, stress response and resistance to pathogen infection. Cloning the sesquiterpene synthase genes of tobacco and studying their function can provide theoretical basis for functional identification and disease resistance breeding of tobacco. In this study, a sesquiterpene synthase gene NtTPS21 was cloned from common tobacco variety Honghuadajinyuan, and its function was identified by bioinformatic analysis, expression pattern analysis, enzyme property analysis and bacteriostatic test in vitro of NtTPS21 fermentation products. The results showed that the open reading frame of NtTPS21 was 1671 bp which encoded 556 amino acids. NtTPS21 had a typical terpene synthase catalytic active site and high homology with Arabidopsis thaliana sesquiterpene synthase AtTPS21. NtTPS21 was localized in the cytoplasm and could be induced by Granville wilt of tobacco. The expression level of NtTPS21 in the resistant variety “Yanyan 97” was significantly higher than that in the susceptible variety “Changbohuang”. NtTPS21 can catalyze the synthesis of sesquiterpene β-caryophyllene, which has a significant inhibitory effect on the growth of granville wilt. These results indicate that NtTPS21 and its metabolites may be related to the resistence to Ralstonia solanacearum.