Abstract: Liquid-liquid phase separation (LLPS) of biomacromolecules is recognized as a key mechanism mediating a wide range of physiological activities in living organisms and has attracted increasing attention in recent years. Phase separation has been observed in various pathological conditions, including neurodegenerative diseases, cancer, viral infections, bacterial infections, and immune-inflammatory diseases, indicating that LLPS has widespread biological functions and potential pathological significance in the occurrence, progression, and regulation of multiple diseases. Based on the underlying mechanisms of LLPS formation, this review focuses on the mechanisms of disease-related aberrant phase separation and systematically summarizes recent studies on the regulation of LLPS, covering the mechanisms of action of various small-molecule drugs and potential candidate compounds. Furthermore, this review discusses the feasibility and prospects of disease intervention and novel drug development through LLPS regulation, aiming to deepen the understanding of LLPS regulatory mechanisms and provide new insights into the development of therapeutic strategies and innovative drugs for related diseases.