The immunological synapse (IS) serves as the fundamental architectural framework for direct interactions and secretory crosstalk between immune cells, as well as between immune cells and other cells. Its dysregulation is thought to be a key underlying cause of immune evasion or inflammation observed in various diseases, including tumors and infections. Numerous recent studies have addressed key signaling mechanisms and reported novel targets related to IS, further broadening our understanding of its function and regulatory factors. However, a comprehensive review that highlights recent progress and consolidates past knowledge is still lacking. In this study, we delineated the pre- and postsynaptic structures constituting the IS between T cells, natural killer (NK) cells, dendritic cells (DCs), and macrophages. We also detail the specific signaling mechanisms and pathways that modulate the formation and disassembly of the IS, including cytoskeletal remodeling, membrane reshaping, integrin signaling, and force transduction. Following these experimental findings, we systematically review the central roles of IS in maintaining homeostasis and health and outline various diseases arising from IS disorders. Finally, we thoroughly explore targets and treatments related to IS on the basis of preclinical evidence and clinical trials, with the aim of providing further investigatory and therapeutic insights for researchers and clinicians.