A targeted nanomedicine with humanized anti-EGFR scFv (NM-scFv) was developed for siRNA delivery into triple negative breast cancer (TNBC) cells. NM-scFv consisted of i) targeted nanovector (NV-scFv): nano-cargo with targeting properties; ii) siRNA: pharmacological agent and iii) cationic polymers (chitosan, poly-L-arginine): for siRNA complexation and endosomal escape. NV-scFv was based on superparamagnetic nanoparticle (SPION) labeled with Dylight™680, a PEG layer and a humanized anti-EGFR scFv. The PEG density was optimized from 236 ± 3 to 873 ± 4 PEGs/NV-scFv and the number of targeting ligands per NV-scFv was increased from 9 to 13. This increase presented a double benefit: i) enhanced cellular internalization by a factor of 2.0 for a 24 h incubation time and ii) few complement protein consumption reflecting a greater stealthiness (26.9 vs 45.3% of protein consumption at 150 µg of iron/mL of NHS). A design of experiments was performed to optimize the charge ratios of chitosan/siRNA (CS) and PLR/siRNA (CR) that influenced significantly: i) siRNA protection and ii) gene silencing effect. With optimal ratios (CS = 10 and CR = 10), anti-GFP siRNA was completely complexed and the transfection efficiency of NM-scFv was 69.4% vs 25.3% for non-targeted NM. These results demonstrated the promising application of our NM-scFv for the targeted siRNA delivery into TNBC cells.