In this study, to improve the efficiency of an aqueous two-phase system (ATPS), a different scenario has been proposed. To this purpose, an endeavor has been undertaken to find a nanoparticle suitable for designing a process of favorable economic separation as well as to unravel the mechanism of the partitioning of nisin along with nanoparticle. First, the capabilities of several different nanoparticles were assessed. These nanoparticles were regarded as additives apt to improve nisin partitioning in the ATPS containing polyethylene glycol (with different molecular weights) and a mixture of potassium hydrogen phosphate and potassium dihydrogen phosphate. The impact of the addition of the modified nanographite (in very small amounts) on the improvement of the nisin partition coefficient in polymer/salt ATPS was quite remarkable. The results showed that the partition coefficient of nisin was increased up to 610% (eight times more than that of the ATPS without nanoparticles) in PEG 10 000 + KH2PO4/K2HPO4 + H2O. The mechanism of the partitioning of nisin along with nanoparticle dominating the boost in the partition coefficient was explained by the structural investigations.