Cell fusion is fundamental to various morphological and physiological events involved in the development of most eukaryotic organisms. Saccharomyces cerevisiae (S. cerevisiae) is a classic model organism for eukaryotic genome synthesis and transfer in the context of synthetic biology. However, the molecular mechanism underlying the yeast cell fusion remains to be fully understood, thereby limiting its synthetic biology application. In S. cerevisiae, mating initiates when cells respond to pheromones that trigger MAPK (mitogen-activated protein kinase) cascade, following with polarization, cell wall remodeling, membrane fusion, and karyogamy. This review discusses the current state of knowledge and progress regarding cell fusion in S. cerevisiae as well as the proteins involved in these events. Especially, the study of the possible “fusase” Prm1 provides a direction for promoting the manipulation of cell fusion. We further propose a hypothesis about the intracellular transport and maturation process of Prm1, which reasonably explains the regulation mechanism of site-directed aggregation of Prm1 at the plasma membrane. Notably, this review stresses the synthetic biology applications of yeast mating signaling pathway in biological components, biological devices and systems, and multicellular interactions. Such elements, including pheromone-responsive promoters, G protein-coupled receptors, scaffold proteins, transcription factors, bistable switches, tuners and chassis cells. Together they contribute to the applications of biosensors and metabolic engineering. Strategies such as rationally engineering of modular circuits and optimizing the reproductive pathway will promote the maneuverability of cell fusion. Moreover, many innovative synthetic biology tools, such as microfluidics, omics research, genome editing, and machine learning, allow researchers to examine the complex physiological activities and improve fusion efficiency. Our study lays foundation for the study of cell-fate decision system and the application of yeast cell fusion in the large genome transfer.