Cargocomplexes play a vital role in non-viral delivery methods due to their capacity to target certain cells (or cells through the cell-division cycle) and inject their (macro)molecular "cargo" into them. The development of gene carriers that can efficiently transport and deliver genetic material into human-targeted cells with minimal toxicity is an important challenge in the field. The present study reports the straightforward preparation and testing of a modular non-viral gene carrier based on AuNPs. The design, synthesis, and in vitro evaluation of multilayer gold nanoparticles (AuNPs) as non-viral gene carriers with high transfection efficiency, reduced cytotoxicity for targeted therapeutic delivery of nucleic acids to MCF-7 cancer cells are presented. The developed non-viral vector is based on supramolecular "host-guest" inclusion complexes of β-cyclodextrin, positioned on the AuNPs surface over a layer of polyethyleneimine, and adamantyl moiety from polyethylene glycol conjugated decapeptide (WXEAAYQRFL). First, the β-CD functionalized PEI was utilized as the template for the synthesis of AuNPs of controlled sizes. The reaction produced small AuNPs with a cationic layer which is known for efficient condensation of genetic material and β-CD suitable for the decoration of the carrier with targeting moieties using "host-guest" inclusion complexation. Subsequently, adamantine-polyethylene glycol conjugated decapeptide was attached to the AuNPs. The in vitro results have validated the ability of the proposed systems to selectively target tumor cells with high efficacy and low toxicity due to the unique affinity of the aptamer-functionalized nanoparticles toward breast cancer cells. The findings of this work demonstrated that the proposed modular system may represent a very promising platform for the AuNP-based non-viral vectors mainly due to the versatility of the system, which allows for the facile exchange of several types of ligands for improving the targeting properties and transfection efficiency, or for providing better protection from the endocytotic systems.