Three-dimensional (3D) tumor has been considered as the best in vitro model for cancer research. In recent years, various methods have been developed to controllable prepare multisize 3D tumors. Nonetheless, reported technologies are still problematic and difficult to produce 3D tumors with highly uniform size and cell content. Here, a novel and simple microsphere-based mold approach is proposed to rapidly fabricate spherical microwell arrays for multisize 3D tumors formation, culture, and recovery. Larger amounts of HepG2 3D tumors with excellent quality and uniformity can be efficiently generated using this method. In addition, the tumor size can also be simply controlled by adjusting the diameter of the microwell arrays. All experimental results indicated that the proposed method offers a promising platform to generate and recover highly controlled multisize 3D tumors for various cell-based biomedical research.