In recent years, 3D integrated microfluidic systems have become increasingly more popular because of their ability to incorporate multifunctional components, including porous membranes and biological scaffolds. Because of limitations in resolution, fabrication efficiency and materials, it is hard to develop complex integrated microfluidic systems with low cost and high efficiency. In this paper, we present a novel method that utilizes modular structure-based design, which could greatly reduce the time and cost for customization of complete integrated chips, compared to traditional techniques. By printing sacrificial patterns on the substrate using the 3D printing approach and subsequently covering them with PDMS prepolymer, PDMS slices with modular structures were obtained, each with specific functions. By combining different PDMS slices with specific modular structures and other functional components, such as membranes and scaffolds, the conceptual design was efficiently converted into complete integrated microfluidic chips. As proof-of-concept, customized 3D microfluidic chips were generated and successfully used for cell culture and biological analysis. Furthermore, the flexible combination with biofabrication of hydrogel beads was also presented, revealing the potential use of this technique in the fabrication of organ-on-a-chip.