Single-cell sequencing (SCS), including genomic, transcriptomic, epigenomic, proteomic, and metabolomic sequencing, is a powerful tool for revealing cellular and molecular landscapes at the level of single-cell resolution. The use of SCS in upper and lower airway diseases has revealed numerous intrinsic biological characteristics and dynamics of airway inflammation, cancer, viral infection, and other lesions, and has also constructed an immune landscape of health and disease states. In this report, we review how advances in SCS technology have increased our understanding of upper and lower airway diseases, including chronic rhinosinusitis, allergic rhinitis, asthma, chronic obstructive pulmonary disease, pulmonary fibrosis, and other inflammatory diseases, as well as the landscape of airway immune cells in healthy and pathological conditions. We also discuss the potential mechanisms of tumor heterogeneity, circulating tumor cells, and tumor biological behavior. The rapid development of single-cell technology will further increase our understanding of the biological characteristics of upper and lower airway diseases and provide precise treatment targets for patients.