The human epidermal growth factor receptor 2 (HER2) is a transmembrane glycoprotein with tyrosine kinase activity involved in different cellular processes such as proliferation, apoptosis, and differentiation. HER2 is overexpressed in several types of solid tumors, including breast and prostate cancer, and it has been associated with aggressiveness, poor prognosis, resistance to therapy, and fast growth. Recent advances in nanomedicine have shown potential for application in cancer detection. In this study, we designed a nanosystem (NS) based on gold nanoparticles (AuNPs) conjugated with a fluorescent HER2-specific aptamer to detect and treat HER2-positive cancer cells. The conjugate formation was characterized by UV-Vis spectroscopy, agarose gel electrophoresis, dynamic light scattering (DLS), and transmission electron microscopy (TEM). Then we evaluated the hemocompatibility and compared the cytotoxic effect between NS and trastuzumab in three cancer cell lines (LNCaP, HCC1954, and ZR7530) and a non-cancerous cell line (Vero CCL-81) as a control. Characterization methods confirmed the conjugation of the fluorescent aptamer to the surface of AuNPs. The hemolysis test showed little to no hemolytic activity of the NS at the concentrations evaluated (0.05, 0.5, and 5 ng/µL). Results of viability assays showed different effects, depending on NS dose and HER2 expression levels between cell lines. Fluorescence detection suggests that NS can specifically bind to cells with high HER2 expression. This preliminary study reveals the potential of aptamer-functionalized AuNPs as a theranostic tool in HER2 overexpressing cancer cells.