Abstract: Liquid-liquid phase separation (LLPS), a novel mechanism of the organization and formation of cellular structures, plays a vital role in regulating cell fate transitions and disease pathogenesis and is gaining widespread attention. LLPS may lead to the assemblage of cellular structures with liquid-like fluidity, such as germ granules, stress granules, and nucleoli, which are classic membraneless organelles. These structures are typically formed through the high-concentration liquid aggregation of biomacromolecules driven by weak multivalent interactions. LLPS is involved in regulating various intracellular life activities and its dysregulation may cause the disruption of cellular functions, thereby contributing to the pathogenesis and development of neurodegenerative diseases, infectious diseases, cancers, etc. Herein, we summarized published findings on the LLPS dynamics of membraneless organelles in physiological and pathological cell fate transition, revealing their crucial roles in cell differentiation, development, and various pathogenic processes. This paper provides a fresh theoretical framework and potential therapeutic targets for LLPS-related studies, opening new avenues for future research.