Three-phase partitioning (TPP) is rapidly developing as a novel bioseparation technique for the separation and purification of biomolecules. TPP has been applied in a wide range of applications including enzyme stability and enhancement of its catalytic activity. The partitioning into three phases is mainly dependent on the concentration of alcohol and salt used. TPP provides high enzyme recovery and can be utilized along with external techniques such as ultrasound, microwave assisted, microaffinity ligand-facilitated and also ionic-liquid based. This technique has attracted interest in the large scale recovery of proteins from crude feedstocks or fermentation broths. In this review, the basic principles, refolding of proteins using TPP, key design variables of TPP, types of TPP, applications of TPP in food industry as well as the challenge of TPP were analyzed. The work presented in this review will be beneficial for further researches in TPP or related separation techniques.