Single-cell analysis is essential to improve our understanding of cell functionality from cellular and subcellular aspects for diagnosis and therapy. Single-cell cultivation is one of the most important processes in single-cell analysis, which allows the monitoring of actual information of individual cells and provides sufficient single-cell clones and cell-derived products for further analysis. The microfluidic device is a fast-rising system that offers efficient, effective, and sensitive single-cell cultivation and real-time single-cell analysis conducted either on-chip or off-chip. Here, we introduce the importance of single-cell cultivation from the aspects of cellular and subcellular studies. We highlight the materials and structures utilized in microfluidic devices for single-cell cultivation. We further discuss biological applications utilizing single-cell cultivation-based microfluidics, such as cellular phenotyping, cell-cell interactions, and omics profiling. Finally, present limitations and future prospects of microfluidics for single-cell cultivation are also discussed.