Three-dimensional multicellular spheroids (MCSs) have received extensive attention in the field of biomedicine due to their ability to simulate the structure and function of tissues in vivo more accurately than traditional in vitro two-dimensional models and to simulate cell-cell and cell extracellular matrix (ECM) interactions. It has become an important in vitro three-dimensional model for tumor research, high-throughput drug screening, tissue engineering, and basic biology research. In the review, we first summarize methods for MCSs generation and their respective advantages and disadvantages and highlight the advances of hydrogel and microfluidic systems in the generation of spheroids. Then, we look at the application of MCSs in cancer research and other aspects. Finally, we discuss the development direction and prospects of MCSs