Pluripotent stem cells are characterized by self-renewal and multi-differentiation potential, which can be used to reverse structurally dysfunctional tissues and organs back to a structurally and functionally intact state of health through repair, replacement, or in-situ regeneration of new cells, tissues, and even organs. Cells or multicellular systems derived from pluripotent stem cell differentiation, especially organoids, have great potential for application in regenerative medicine. However, the clinical application of stem cell-related therapies is still in its infancy, and the current challenges to the clinical translation of stem cells include the tumorigenicity, heterogeneity, and immunogenicity of stem cell derivatives. Synthetic biology, with its “top-down” design concept and powerful toolkit including synthetic receptors and gene circuits, allows for the rational assembly of standardized modules. With the rapid development of gene editing technology and the deepening of cell biology research, the engineering object of synthetic biology has shifted from lower model organisms such as Escherichia coli or Saccharomyces cerevisiae to mammalian cells. On the one hand, “top-down” design strategies can engineer stem cells by giving them new functions, and on the other hand, the acquisition of new phenotypes by stem cells can test known gene functions and improve understanding of cell biology. Therefore, the application of these synthetic biology tools to stem cell engineering provides new strategies and platforms for relevant cell therapies or organ transplantation. It offers potential advantages in precise control of cell fate, regulation of cell communication, optimization of organoid structure and function, and monitoring and elimination of tumorigenic cells. These synthetic biology tools have provided new strategies and platforms for the engineering and reprogramming of stem cells, offering the potential to address current challenges in the clinical application of stem cells. They are expected to drive further advancements in regenerative medicine and ultimately achieve the core goal of regenerative medicine, which is organ regeneration.