Adenoviral vectors are crucial for gene therapy and vaccine development, offering a platform for gene delivery into host cells. Since the discovery of adenoviruses, first-generation vectors with limited capacity have evolved to third-generation vectors flacking viral coding sequences, balancing safety and gene-carrying capacity. The applications of adenoviral vectors for gene therapy and anti-viral treatments have expanded through the use of in vitro ligation and homologous recombination, along with gene editing advancements such as CRISPR-Cas9. Current research aims to maintain the efficacy and safety of adenoviral vectors by addressing challenges such as pre-existing immunity against adenoviral vectors and developing new adenoviral vectors from rare adenovirus types and non-human species. In summary, adenoviral vectors have great potential in gene therapy and vaccine development. Through continuous research and technological advancements, these vectors are expected to lead to the development of safer and more effective treatments.