Abstract: UraniumU(Ⅵ) in the blood is known to form stable complexes with apotransferrin (apo-Tf), which plays an important role in mediating the cytotoxicity induced by U(Ⅵ) transported to cells. The present study aimed to establish an new in vitro screening model of U(Ⅵ) decorporation agents through exploring the capability of chelating agents competing with U(Ⅵ) binding to apo-transferrin based on enzyme-linked immunosorbent assay (ELISA). The optimal concentrations of apo-Tf coated antigen, Tf antibody, secondary antibody and U(Ⅵ) treatment were achieved and the stability and reproducibility of this method were validated by methodology study. Using this model, the ability of four chelating agents to mobilize the U(Ⅵ) binding to apo-Tf was evaluated, and the rank of competitiveness was catechol-3,6-bis(methyleiminodiacetic acid) (CBMIDA) ≈ Tiron > apo-Tf > DTPA-CaNa₃ ≈ DTPA-ZnNa₃. The efficacy of these chelating agents in removal of U(Ⅵ) was tested by animal experiments. The results showed that immediate administration of CBMIDA or Tiron after injection of U(Ⅵ) in mice significantly promoted urinary U(Ⅵ) excretion and reduced U(Ⅵ) accumulation in kidneys and femurs, while DTPA-CaNa₃ and DTPA-ZnNa₃ have no obvious effects as compared to U(Ⅵ)-exposed mice alone, which was consistent with the results of competitive ELISA method. The animal experiments conform to the rules of the Animal Research Ethics Committee of School of Pharmacy of Fudan University. These results show that the new proposed method is rapid, simple and convenient with good reproducibility and has the potential to be used for in vitro screening of U(Ⅵ) decorporation agents.