The safety issues like immunogenicity and unacceptable cancer risk of viral vectors for DNA/mRNA vaccine delivery necessitate the development of non-viral vectors with no toxicity. Among the non-viral strategies, cell-penetrating peptides (CPPs) have been a topic of interest recently because of their ability to cross plasma membranes and facilitate nucleic acids delivery both in vivo and in vitro. In addition to the application in the field of gene vaccine and gene therapy, CPPs based nucleic acids delivery have been proved by its potential application like gene editing, RNA-sequencing, and imaging. Here, we focus on summarizing the recent applications and progress of CPPs-mediated nucleic acids delivery and discuss the current problems and solutions in this field.