Establishing an in vitro–based cell culture system that can realistically simulate in vivo cell dynamics is desirable. It is thus necessary to develop a method for producing a large amount of cell aggregates (i.e., spheroids) that are uniform in size and quality. Various methods have been proposed for the preparation of spheroids; however, none of them satisfy all requirements, such as cost, size uniformity, and throughput. Herein, we successfully developed a new cell culture method by combining fluoropolymers and dot patterned extracellular matrix substrates to achieve size-controlled spheroids. First, the spheroids were spontaneously formed by culturing them two-dimensionally, after which the cells were detached with a weak liquid flow and cultured in suspension without enzyme treatment. Stable quality spheroids were easily produced, and it is expected that the introduction and running costs of the technique will be low; therefore, this method shows potential for application in the field of regenerative medicine.