Small interfering RNA (siRNA) highlights the immense therapeutic potential for cancer treatment. The major challenge in siRNA therapy is the effective RNA nanodrug delivery system, which is facilitated by the ligand and the carrier. In this study, we analyzed the binding specificity of linear RGD and circular RGD to αVβ3 integrins by mapping the morphology using super-resolution direct stochastic optical reconstruction microscopy. Meanwhile, the binding dynamics was investigated using single-molecule force spectroscopy. Then, the effects of the ligand and carrier on RNA nanodrug cell entry dynamic parameters were evaluated at the single particle level by the force tracing technique. Furthermore, the delivery efficiency of RNA nanodrugs was assessed using AFM-based nanoindentation at the single cell level. This report will provide valuable insights for rational design strategies aiming to achieve improved efficiency for nanodrug delivery systems.