Tobacco (Nicotiana tabacum, 2n = 48) is a key non-food economic crop, yet its stress response and gene regulatory mechanisms remain poorly understood. By analyzing 603 transcriptome datasets, this study identified 1405 tissue-specific genes, revealing tissue-specific synthesis of terpenoids and other ecologically important secondary metabolites in sepals and other tissues. Comparative stress-response analysis highlighted distinct gene expression patterns in leaves and roots under biotic and abiotic stresses. Additionally, 28,396 expression quantitative trait loci (eQTLs) were mapped in leaves, offering valuable genetic regulatory markers. These findings provide crucial insights into tobacco’s gene expression characteristics and their functional implications, serving as a foundation for future research.