Spheroids are expected to aid the establishment of an in vitro-based cell culture system that can realistically reproduce cellular dynamics in vivo. We developed a fluoropolymer scaffold with an extracellular matrix (ECM) dot array and confirmed the possibility of mass-producing spheroids with uniform dimensions. Controlling the quality of ECM dots is important as it ensures spheroid uniformity, but issues such as pattern deviation and ECM drying persist in the conventional microstamping method. In this study, these problems were overcome via ECM dot printing using a resin mask with dot-patterned holes. For dot diameters of φ 300 μm, 400 μm, and 600 μm, the average spheroid diameters of human iPS cells (hiPSCs) were φ 260.8 μm, 292.4 μm, and 330.7 μm, respectively. The standard deviation when each average was normalized to 100 was 14.1%. A high throughput of 89.9% for colony formation rate to the number of dots and 89.3% for spheroid collection rate was achieved. The cells proliferated on ECM dots, and the colonies could be naturally detached from the scaffold without the use of enzymes, so there was almost no stimulation of the cells. Thus, the undifferentiated nature of hiPSCs was maintained until day 4. Therefore, this method is expected to be useful in drug discovery and regenerative medicine.