The liquid–liquid phase separation (LLPS) of biomolecules induces condensed assemblies called liquid droplets or membrane-less organelles. In contrast to organelles with lipid membrane barriers, the liquid droplets induced by LLPS do not have distinct barriers (lipid bilayer). Biomolecular LLPS in cells has attracted considerable attention in broad research fields from cellular biology to soft matter physics. The physical and chemical properties of LLPS exert a variety of functions in living cells: activating and deactivating biomolecules involving enzymes; controlling the localization, condensation, and concentration of biomolecules; the filtration and purification of biomolecules; and sensing environmental factors for fast, adaptive, and reversible responses. The versatility of LLPS plays an essential role in various biological processes, such as controlling the central dogma and the onset mechanism of pathological diseases. Moreover, biomolecular LLPS could be critical for developing new biotechnologies such as the condensation, purification, and activation of a series of biomolecules. In this review article, we introduce some fundamental aspects and recent progress of biomolecular LLPS in living cells and test tubes. Then, we discuss applications of biomolecular LLPS toward biotechnologies.