A well-known bioseparation technique namely liquid biphasic system (LBS) has attracted many researchers' interest for being an alternative bioseparation technology for various kinds of biomolecules. The present review begins with an in-depth discussion on the fundamental principle of LBS and this is followed by the discussion on further development of various phase-forming components in LBS. Additionally, the implementation of various advance technologies to the LBS that is beneficial towards the efficiency of LBS for the extraction, separation, and purification of biomolecules was discussed. The key parameters affecting the LBS were presented and evaluated. Moreover, future prospect and challenges were highlighted to be a useful guide for future development of LBS. The efforts presented in this review will provide an insight for future researches in liquid-liquid separation techniques. © 2020 by the authors.