In recent years, a big trend has been the development of rapid, green, efficient, economical, and scalable approaches for the separation and purification of bioactive molecules from natural sources, which can be used in food, cosmetics, and medicine. As a new nonchromatographic bioseparation technology, three-phase partitioning (TPP) is attracting the attention of a growing number of scientists and engineers. Although a number of studies have been published in the last 40 years regarding the extraction, separation, and purification of numerous bioactive molecules using TPP systems, a background review on TPP partitioning fundamentals and its applications is much needed. Therefore, the present review focuses in detail on the TPP separation process, including the definition of TPP, partitioning mechanisms, parameters for establishing the suitable condition to form precipitate such as concentration of ammonium sulfate, content of tert-butanol, pH and temperature, and the application for separation and purification of protein, enzyme, plant oil, polysaccharide, and other small molecule organic compounds. In addition, the possible directions of future developments in TPP technology are discussed. The review presents a good opportunity, as well as a challenge for scientists, to understand the detailed partitioning rule and to take better use of TPP for the production and separation of various bioactive molecules, which have been intensively applied in the food and medical fields.