Leaf glandular trichomes of Nicotiana tabacum represent a biologically active and stress-responsive tissue that contributes to plant defence response against biotic and abiotic stress and also influences leaf aroma and smoke flavour. Although a number of research groups have been focusing on developmental aspects of glandular trichome biology for several decades, the understanding about the development of glandular trichomes is still quite fragmentary. Single-cell RNA sequencing (scRNA-seq) technology is a powerful tool for discovering heterogeneous cells and development or differentiation of specific cell types. To explore the developmental trajectory of glandular trichomes, we extracted single nuclei from young tobacco leaves and successfully captured > 15,000 nuclei for single-nucleus sequencing (snRNA-seq). After cell type annotation using Arabidopsis homologous gene, all nuclei were divided into three large groups, mesophyll, epidermis and vasculature. The cells belonging to glandular trichomes were further identified by GO enrichment analysis and expression patterns of glandular trichomes-specific genes and terpenoid biosynthesis-related genes. At the same time, cell trajectory analysis was performed to delineate the developmental trajectory of glandular trichomes. Our results provide a good case for studying development of tobacco glandular trichomes, and the development-related genes identified by this study also provide a valuable resource for tobacco breeding.