Breast cancer remains a challenging health issue, demanding innovative treatment approaches that maximize efficacy while minimizing damage to healthy cells. Targeted therapy offers a promising strategy tailored to the unique characteristics of breast cancer tumors. Gold nanoparticles have been studied in the context of their therapeutic potential towards cancer treatment showing great success. Recently, aptamers were also investigated for their targeting efficiency towards specific receptors allowing their use in targeting delivery systems. In this study, computational analysis was used to confirm the strong binding between AS1411 aptamer and the nucleolin receptor extensively present on the surface of breast cancer cells, highlighting the aptamer’s potential for specific targeting. Furthermore, we investigated and compared the use of AS1411 aptamer-conjugated chemically synthesized (GNPs) and flaxseed-green-synthesized (Fs-GNPs) gold nanoparticles as targeting therapeutic systems for breast cancer cells. Our results showed successful conjugation of the AS1411 aptamer with both, the GNPs and Fs-GNPs. Characterization of the nanoparticles and their conjugates validates their size, charge, and morphology, affirming the success of the conjugation process. Cytotoxicity assessments using the MTT assay demonstrated the effectiveness of the conjugates against breast cancer cells, with the AS1411-Fs-GNPs conjugate exhibiting higher inhibitory efficacy, featuring an IC50 value of 11.13 µg/ml. In contrast, they showed minimal effect on normal cells, emphasizing the selectivity and potential safety of these therapies. To our knowledge, this is the first report of conjugating AS1411 aptamer to green-synthesized gold nanoparticles and its use as a targeting therapeutic system.