Abstract: Currently, malignant tumors are a major public health problem and pose a serious threat to human health. Because of the diversity and complexity and the high mortality rates, they have been a popular but complicated research topic for several years. Heterogeneity is one of the main characteristics of malignant tumors, and thus, this characteristic may affect many other aspects, such as tumorigenesis, development, metastasis, and therapeutic effects. With the completion of the Human Genome Project and the continuous advancement in sequencing technology, single-cell sequencing technology has been applied in many medical research fields; in particular, it is useful for analyzing tumor cell subpopulations, cell heterogeneity, tumor occurrence, evolution, and drug resistance. Therefore, this technology has provided us with a new tool for clinical diagnosis and treatment of malignant tumors and for estimating the prognosis at an early stage. In this article, we aimed to review the advances in single-cell sequencing technology and its application in malignant tumor research.