A stable, biocompatible and exquisite SPIONs-PEG-HER targeting complex was developed. Initially synthesized superparamagnetic iron oxide nanoparticles (SPIONs) were silanized using 3-aminopropyltrimethoxysilane (APS) as the coupling agent in order to allow the covalent bonding of polyethylene glycol (PEG) to the SPIONs to improve the biocompatibility of the SPIONs. SPIONs-PEG were then conjugated with herceptin (HER) to permit the SPIONs-PEG-HER to target the specific receptors expressed over the surface of the HER2+ metastatic breast cancer cells. Each preparation step was physico-chemically analyzed and characterized by a number of analytical methods including AAS, FTIR spectroscopy, XRD, FESEM, TEM, DLS and VSM. The biocompatibility of SPIONs-PEG-HER was evaluated in vitro on HSF-1184 (human skin fibroblast cells), SK-BR-3 (human breast cancer cells, HER+), MDA-MB-231 (human breast cancer cells, HER-) and MDA-MB-468 (human breast cancer cells, HER-) cell lines by performing MTT and trypan blue assays. The hemolysis analysis results of the SPIONs-PEG-HER and SPIONs-PEG did not indicate any sign of lysis while in contact with erythrocytes. Additionally, there were no morphological changes seen in RBCs after incubation with SPIONs-PEG-HER and SPIONs-PEG under a light microscope. The qualitative and quantitative in vitro targeting studies confirmed the high level of SPION-PEG-HER binding to SK-BR-3 (HER2+ metastatic breast cancer cells). Thus, the results reflected that the SPIONs-PEG-HER can be chosen as a favorable biomaterial for biomedical applications, chiefly magnetic hyperthermia, in the future.