Aqueous two-phase system (ATPS) is a system composed of two incompatible solutions, which has excellent performance for extracting proteins, antibiotics, and other bioactive molecules, so it is a very simple, efficient, and green extraction and separation method. In this paper, we introduced three different emerging micromolecule-based ATPSs and introduced their characteristics, applications, advantages, and disadvantages respectively. This is of guiding significance for the synthesis of new ATPSs. One of the important prospects of this ATPS in industrial application is the separation and purification of fermentation-based biofuel. Adding a salt, sugar, or ionic liquid to a biofuel solution can remove most of the water, enabling efficient recovery and dehydration of biofuel. The two-step process can replace the traditional distillation and molecular sieve adsorption processes to obtain high-purity bioalcohols, thus introducing a new purification process for biofuel. An in-depth analysis of the separation performance of the emerging micromolecule-based ATPSs as well as an objective indication of their application provides valuable insights for further optimization and application of this kind of ATPSs in separation science.