Abstract: Flow cytometry is a rapid and sensitive technology in cell analysis and sorting. At present, it has been widely used in many fields of plant research. In order to explore the application of flow cytometry in tobacco research, this article described the working principle of flow cytometry, and reviewed the current application of flow cytometry in plant research, such as DNA content in plant nuclear and ploidy analysis, chromosomal karyotype analysis and sorting, cell sorting and integrating with other technologies. Furthermore, the applications of flow cytometry in tobacco research were reviewed, and the possibility of further application in tobacco single-cell omics, disease control and cigarette smoke toxicology detection was prospected.