Cell-penetrating peptides (CPPs) have gained prominence in cellular drug delivery due to their extremely low toxicity and rapid cell internalization properties. Understanding the effect of CPPs' physicochemical properties on trans-membrane behavior will provide a better screening scheme for designing effective CPP-conjugated nano-drugs. Herein, the efficiency of the CPPs interacting with the cell membrane and the subsequent trans-membrane is revealed at the single-molecule level using single-molecule force spectroscopy (SMFS) and force tracing technique based on atomic force spectroscopy (AFM). The dynamic force spectroscopy (DFS) analysis indicates that cationic TAT48-60 and amphipathic MAP are more effective during the interaction with cell membrane due to the strong electrostatic interaction between CPPs and cell membrane. However, for the subsequent trans-membrane process, the hydrophobicity of Pep-7 plays a key role, showing a higher trans-membrane speed at the single-molecule level. Meanwhile, Pep-7 shows lower trans-membrane speed and probability on normal cells (Vero), which makes it more suitable as a peptide-based nano-drug to treat tumors avoiding harming normal cells. The dynamic parameters obtained in this study offer guidance for screening and modifying effective CPPs, targeting specific cell lines or tissues during the nano-drug design.