In order to reveal the events during the course of killing target cells of immunotoxins, two forms of Pseudomonas exotoxin A based immunotoxins against carcinoembryonic antigen (CEA) were constructed and named CEA(Fv)/PE38/KDEL and PE35/CEA(Fv)/KDEL. Antigen binding activity of the immunotoxins were assessed by flow cytometry, internalization of immunotoxins was detected by optical confocal microscopy and flow cytometry quantitatively. Cell apoptosis and necrosis were analyzed by staining the cells with FITC-annexin V/PI. The MTT method was used to assay the cytotoxicity of the immunotoxins. The biological functions of the two forms of immunotoxins in the different functional phases were compared. And the interaction between different functional phases was analyzed. The results revealed the whole functional process from the cell binding to the cytotoxicity of the immunotoxins, which provided more details about the biological functions of immunotoxins and could be valuable in the mechanism research and the optimization of immunotoxins.