Three-dimensional cell culture systems offer greater understanding of the complex human body structure than monolayer cell cultures. Spheroids, which are the most useful and controllable types of three-dimensional cell formations, are discussed in this review. Conventional spheroid fabrication methods have limitations for the mass production of uniformly sized spheroids, which hinders their further application. As an alternative, microfabrication methods have been proposed to overcome the drawbacks of existing methods. Microfabrication technologies include micropatterning, 3D bioprinting, and microfluidics. Microwell arrays and surface-modified micropatterns can be fabricated through micropatterning methods, and these scaffolds result in the mass production of spheroids with size uniformity. 3D bioprinting technology enables uniformly sized spheroid production at desired locations, and microfluidics allows production of uniform size-controlled spheroids in a large quantity. Recently, efforts have been made to apply 3D spheroid culture systems to regenerative medicine, the study of the tumor microenvironment, drug screening, and organoid fabrication. The 3D spheroid system is an attractive substitute for overcoming the limitations of the conventional 2D culture platform, which cannot precisely imitate in vivo physiological environments. Microfabrication methods for spheroids enhance the effectiveness of spheroid formation, allowing for mass production, size control, and spheroid localization. Microfabrication methods have remarkable potential for spheroid utilization in the biomedical field.