OBJECTIVES: Superparamagnetic iron oxide nanoparticles (SPIONs) have been considered promising non-invasive imaging tools in medicine. However, their high surface energy leads to NPs aggregation, while non-targeted SPIONs can cause cytotoxic effects on normal cells. In this work, we evaluated the potential of polyethyleneimine (PEI)-SPIONs targeted by PNC-27 peptide as a double targeting agent throughout early cancer diagnosis. MATERIALS AND METHODS: Initially, PEI was conjugated to PNC-27 with HDM-2-binding domain. Then, SPIONs were loaded into PEI-PNC-27 through the ligand exchange method. The physicochemical characteristics of the synthesized NPs were evaluated. The cytotoxicity and targeting efficiency were assayed against HT-29 and CT-26 cell lines along with NIH-3t3 as normal cells by MTT method and Prussian blue staining test, respectively. RESULTS: The mean diameter of synthesized carriers was obtained in the range of 86.6 - 116.1 nm with a positive charge. According to the cytotoxicity results, the binding and uptake abilities of the PNC-27 peptide by cancer cells were significantly higher than that of the NIH-3t3 cells. However, the results were indicative of the more toxic impacts of targeted synthesized NPs against CT-26 cancer cell line when being compared with HT-29 cells, which may be caused by the different cytotoxicity mechanisms of NPs. In addition, the targeted carriers and SPIONs were present inside and around the cells with HDM-2 expression along with only a few non-targeted vectors, while displaying no appearance throughout the normal cell. CONCLUSION: The results indicated the efficiency of targeted PEI-coated SPIONs for cancer diagnostic applications.