Abstract: Liquid–liquid phase separation (LLPS) of biomacromolecules is recognized as a key mechanism underlying a wide range of physiological processes in living systems and has attracted increasing attention in recent years. Emerging evidence indicates that multiple amyloid proteins associated with neurodegenerative diseases are capable of undergoing LLPS, and that this phase separation behavior is closely linked to their respective pathogenic mechanisms. In addition, LLPS phenomena have been observed in diverse pathological contexts, including cancer, viral infections, and bacterial diseases, highlighting the widespread biological significance and potential pathological relevance of LLPS in disease initiation, progression, and regulation. In this review, we summarize the mechanisms governing LLPS formation and recent advances in disease-related LLPS research. We further provide a comprehensive overview of representative studies that modulate LLPS processes, with a particular focus on the mechanisms of action of small-molecule drugs and potential candidate compounds. Furthermore, we discuss the feasibility and prospects of targeting LLPS for disease intervention and novel drug development, aiming to deepen the understanding of LLPS regulation and to offer new insights into therapeutic strategy development and innovative drug discovery.