Unique characteristics, such as nontoxicity and rapid cellular internalization, allow the cell-penetrating peptides (CPPs) to transport hydrophilic macromolecules into cells, thus, enabling them to execute biological functions. However, some CPPs have limitations due to nonspecificity and easy proteolysis. To overcome such defects, the CPP amino acid sequence can be modified, replaced, and reconstructed for optimization. CPPs can also be used in combination with other drug vectors, fused with their preponderances to create novel multifunctional drug-delivery systems that increase the stability during blood circulation, and also develop novel preparations capable of targeted delivery, along with sustainable and controllable release. Further improvements in CPP structure can facilitate the penetration of macromolecules into diverse biomembrane structures, such as the blood brain barrier, gastroenteric mucosa, and skin dermis. The ability of CPP to act as transmembrane vectors improves the clinical application of some biomolecules to treat central nervous system diseases, increase oral bioavailability, and develop percutaneous-delivery dosage form.