Cell penetrating peptide (CPP) is a kind of small peptide with the capability of penetrating through cell membrane, which provides efficient tools for achieving intracellular access for macromolecular drugs. At present, it has obtained widespread attention and extensive research. Although it has been found recently that many problems hinder the process of its application research and development, such as drug release rate, metabolic degradation, cell lines and differentiation status and Rho GTPases activity dependence，lots of progress has been made in the research of CPPs in cancer treatment, antibacterial and anti-inflammatory targeted drugs, some of which even has entered the stage of preclinical and clinical research. In this article, we focus on two aspects, the challenges and new opportunities in the studies of CPP application, to summarize the recent research progress, and highlight the prospect of new fields of CPP application in the future.