Abstract: Cell-penetrating peptides (CPPs), such as TAT and L17E, have demonstrated significant potential in drug delivery, particularly in protein delivery. Previous studies have shown that TAT fusion proteins exhibit superior delivery efficiency compared to non-fusion counterparts and under non-covalent conjugation conditions, L17E demonstrates superior delivery efficiency compared to TAT in transporting EGFP into cells. Therefore, this study investigates the ability of TAT and L17E to deliver target proteins via covalent fusion strategies, along with their molecular-level mechanisms of action. The fusion proteins TAT-EGFP and L17E-EGFP were expressed and purified. They were evaluated for their abilities to penetrate B16-F10 cells using flow cytometry and fluorescence microscopy. The results revealed that TAT-EGFP exhibited stronger membrane-penetrating ability, with delivery efficiency positively correlated with fusion protein concentration. Structural simulations using AlphaFold3 indicated that TAT-EGFP possesses two outward-extending hydrophobic side chains, a structural feature likely enhancing its interaction with the cell membrane and thereby improving penetration efficiency. In contrast, L17E-EGFP displayed inward-retracted side chains and intramolecular electrostatic interactions, which may reduce binding sites and contact area with the cell membrane, impairing penetration efficacy. This study demonstrates that the structural characteristics of cationic cell-penetrating peptides, such as TAT and L17E (including side chain conformation, hydrophobicity, and charge distribution) are critical determinants of their delivery efficiency, providing a pivotal foundation for optimizing CPP design and advancing CPP-mediated protein drug development.