ABSTRACTA tumor’s heterogeneity has important implications in terms of its clonal origin, progression, stemness, and drug resistance. Therefore, because of its significance in treatment, it is important to understand the gene expression pattern of a single cell, track gene expression or mutation in heterogeneous cells, evaluate the clonal origin of cancer cells, and determine the selective evolution of different subpopulations of cancer cells. Researchers are able to trace a cell’s mutation and identify different types of tumor cells by measuring the whole transcriptome with single-cell sequencing (scRNA-seq). This technology provides a better understanding of the molecular mechanisms driving tumor growth than that offered by traditional RNA sequencing methods. In addition, it has revealed changes in the mutations and functions of somatic cells as a tumor evolves; it has also clarified immune cell infiltration and activation. Research on scRNA-seq technology has recently advanced significantly, suggesting new strategies for the treatment of cancer. In short, cancer researchers have become increasingly dependent on scRNA-seq. This paper reviews the development, detection principles, and processes of scRNA-seq technology and their application in tumor research. It also considers potential clinical applications.