Abstract The content of terpenoids in tobacco can alter its resistance to TMV. NtSPS1, a pivotal structural gene in tobacco, is capable to regulate the terpenoid content. In this study, we investigated the effect of NtSPS1 knockout in HD on the content of terpenoids and the anti-TMV activity of this mutant using gene editing, widely targeted metabolomics, network pharmacology, and molecular docking. 48 terpenoids (six up-regulated and five down-regulated) in NtSPS1 knockout tobacco compared with WT leaves. Notably, solanesol was remarkable downregulation which was lowered by fourfold and compounds 1 (log2FC = 18.2), 8 (log2FC = 16.7) were significant upregulation between the mutants and wild-type line leaves. The 46 terpenoid’s target network encompassed 150 nodes, 509 edges and their underlying mechanisms in the therapeutic management of TMV are discussed. Furthermore, the network pharmacology and molecular docking revealed that compounds 16, 18, 23, 27, and 36 exhibited significant affinity in their respective interactions. Ultimately, five compounds were assayed for their anti-TMV effects, noteworthily, compounds 36 showed potential anti-TMV activity. Above all, we adopted a multifaceted approach to gain a comprehensive understanding of the terpenoid content and anti-TMV properties in NtSPS1 knockout HD. It enlightens the therapeutic potential of NtSPS1 knockout tobacco and it is helpful to find undescribed anti-TMV activity inhibitors, as well as searching for new anti-TMV candidates from the mutants.