Aqueous two-phase systems (ATPS) are a promising biotechnology in bioseparation. However, recycling of the phase-forming polymer is a key issue. In this research, new recyclable ATPS composed of two pH-responsive polymers (P-ADB4.01 and P-MDBH3.27) were prepared. The ATPS could be recycled by adjusting the pH of the solution, with recovery of 93.8%. The ATPS were evaluated for the first time in the partitioning of tylosin. The effects of the main parameters, such as the polymer concentration, pH, temperature, salt species and concentrations, on the partition of crude tylosin and tylosin from fermentation broth were studied. In the 2% P-MDBH3.27/3% P-ADB4.01 ATPS, the partition coefficient (K) and extraction recovery (ERtop) of crude tylosin could reach 5.12 and 93.9%, respectively, in the presence of 90 mM KCl at pH 5.83 and 25 degrees C. In the 2.5% P-MDBH3.27/3% P-ADB4.01 ATPS, the K and ERtop of tylosin from fermentation broth reached 3.58 and 87.3%, respectively, in the presence of 20 mM KBr at pH 5.69 and 25 degrees C. Through partition mechanism analysis by Fourier transform infrared spectroscopy (FT-IR) and interphase potential measurements, the appearance of a new absorption peak and interphase potential difference, respectively, may contribute to the partitioning of tylosin in the P-ADB4.01-enriched top phase.