Single-cell RNA sequencing (scRNA-seq) is a powerful technique that quantifies gene expression at the single-cell level, enabling the elucidation of cellular heterogeneity in organ development and disease. Organoids, serving as miniature in vitro models that mimic the architecture and function of human organs, have emerged as invaluable tools for investigating organ development, disease mechanisms, and drug screening. Traditional organoid research methods often fall short in accurately characterizing cellular heterogeneity, however, the integration of single-cell transcriptome sequencing technology provides unprecedented insights into the gene expression profiles and dynamic changes of distinct cell types within organoids at single-cell resolution. By applying scRNA-seq to organoid models, the resulting data enhance our understanding of intracellular and intercellular biological processes, expand cell transcriptional profiles, and facilitate the identification of specific cell types. Combining scRNA-seq with organoid models has significantly propelled advancements in disease mechanism research, drug screening, and regenerative medicine, offering novel strategies and perspectives for personalized medicine and precision therapy. In this review, we elaborate the specific applications and advantages of scRNA-seq in organoid models, and explores future directions, opportunities, and challenges associated with single-cell transcriptome sequencing technology. It is anticipated that in the near future, the potential of scRNA-seq in organoid research will be further harnessed, thereby broadening the horizons of biomedical research.