Cancer is an intricate disease with inherent intra-tumor heterogeneity at the cellular level because of genetic changes and environmental differences. Cellular heterogeneity exists even within the same tumor type. Small deviations in a genome or transcriptome can lead to significant differences in function. Conventional bulk population sequencing, which produces admixed populations of cells, can only provide an average expression signal for one cell population, ignoring differences between individual cells. Important advances in sequencing have been made in recent years. Single cell sequencing starts in a single cell, thereby increasing our capability to characterize intratumor heterogeneity. This technology has been used to analyze genetic variation, specific metabolic activity, and evolutionary processes in tumors, which may help us understand tumor occurrence and development and improve our understanding of the tumor microenvironment. In addition, it provides a theoretical basis for the development of clinical treatments, especially for personalized medicine. In this article, we briefly introduce Single cell sequencing technology, summarize the application of Single cell sequencing to study the tumor microenvironment, as well as its therapeutic application in different clinical procedures.