Single-cell RNA sequencing technology (scRNA-seq) has been steadily developing since its inception in 2009. Unlike bulk RNA-seq, scRNA-seq identifies the heterogeneity of tissue cells and reveals gene expression changes in individual cells at the microscopic level. Here, we review the development of scRNA-seq, which has gone through iterations of reverse transcription, in vitro transcription, smart-seq, drop-seq, 10 × Genomics, and spatial single-cell transcriptome technologies. The technology of 10 × Genomics has been widely applied in medicine and biology, producing rich research results. Furthermore, this review presents a summary of the analytical process for single-cell transcriptome data and its integration with other omics analyses, including genomes, epigenomes, proteomes, and metabolomics. The single-cell transcriptome has a wide range of applications in biology and medicine. This review analyzes the applications of scRNA-seq in cancer, stem cell research, developmental biology, microbiology, and other fields. In essence, scRNA-seq provides a means of elucidating gene expression patterns in single cells, thereby offering a valuable tool for scientific research. Nevertheless, the current single-cell transcriptome technology is still imperfect, and this review identifies its shortcomings and anticipates future developments. The objective of this review is to facilitate a deeper comprehension of scRNA-seq technology and its applications in biological and medical research, as well as to identify avenues for its future development in alignment with practical needs.