Immunotoxin therapy is one of the immunotherapy strategies providing a new, effective and high potency treatment against various cancers. Breast cancer is the most common cancer among women in many countries. The EPH receptors are a large part of tyrosine kinase receptors family and play an effective role in tumor development and angiogenesis. Among EPH receptors, EPHA2 is more commonly well-known and widely expressed in many cancers like breast cancer. In this study, we evaluated the specification of a designed immunotoxin formed by EPHA2-specific scfv linked with PE38KDEL on EPHA2-overexpressing breast cancer cell line. This new scfv-based recombinant immunotoxin was studied in terms of features such as binding potency, cytotoxicity effects, apoptosis induction ability, and internalization. The flow cytometry results showed that the immunotoxin can significantly (approximately 99%) bind to EPHA2-overexpressing breast cancer cell line (MDA-MB-231) in a low concentration (2.5 ng/ul) while cannot significantly bind to the normal cell line (HEK-293) or even EPHA2-very low expressing cell line (MCF-7). Using the MTT assay and Annexin V/Propidium iodide (PI) double staining method by flow cytometry, we observed significant killing and apoptosis induction of the MDA-MB-231 cells at different concentrations. Immunotoxin tracking by confocal microscopy at 2 h and 6 h revealed a massive presence of immunotoxin in the cytoplasm. Finally, given the in vitro results, it seems that this immunotoxin is competent enough to serve as a good candidate for in vivo studies to further explore the possibility of breast cancer treatment.