The early detection of cancer is very important for the successful treatment of this dangerous disease. The immunomagnetic separation of circulating tumor cells is one of the recent and promising methods for this aim. In the present study, the potential of aptamer targeted iron oxide nanoparticles (IONPs) for the specific and efficient capture of cancer cells was investigated. For this purpose, IONPs were simply synthesized by the solvothermal method and functionalized by a silica layer (FIONPs) and finally targeted with the EpCAM aptamer (EpCAM-FIONPs) via the glutaraldehyde as a linker. The physicochemical characterization by SEM, EDX, FTIR, VSM, XRD and Zetasizer, confirmed the synthesis of colloidal, monodispersed, semi-spherical and crystalline IONPs with the average size of 28.26 nm with the high saturation magnetization value of 38.82 emu g− 1 that were coated by a silica layer and targeted with the EpCAM aptamers. The in vitro MTT assay showed high biocompatibility of EpCAM-FIONPs on the MDA-MB-231 and MCF-7 breast cancer cells. The in vitro studies also showed significant capture efficiency for breast cancer cells such that the highest capture efficiency of 78 ± 2% and 81 ± 3% obtained for MCF-7 and MDA-MB-231 cells, respectively, in the presence of 200 µg mL− 1 EpCAM-FIONPs. The overall results indicated high potential of EpCAM-FIONPs for the detection and isolation of breast cancer cells.