Objective：To investigate the mechanism of endosomal sorting by constructing the transferrin receptor-ubiquitin（TfR-Ubi）protein. Methods：Mouse ubiquitin and human transferrin receptor genes were amplified to construct HA-TfR-Ubi plasmid. Then the expression of HA-TfR-Ubi protein was confirmed by Western blot following by transfected into A431 cells. The A431 and Hrs-KO MEF cells stably expressing HA-TfR-Ubi were treated with transferrin，and the location of HA-TfR-Ubi fusion protein was observed in the cells by immunofluorescence. Results：pHA-TfR-Ubi was successfully constructed. Immunofluorescence results showed that HA-TfR-Ubi fusion protein was sorted into late endosome /lysosomal，and its endosomal sorting was affected in Hrs-KO MEF cells. Conclusion：The endosomal sorting of TfR-Ubi fusion protein demonstrated that the ubiquitin label changed the transferrin receptor degradative pathway. TfR-Ubi plasmid provided a useful experimental tool for the study of ESCRT sorting mechanism.