Abstract：Though there are still many challenges to overcome, gene therapy has led to high expectation for curing a broad range of diseases, especially on the treatment of cancer. One of the key problems in clinical application is to construct safe and efficient gene transfer systems. Replication-defective adenoviruses are main vectors of choice, and many kinds of adenovirus vectors have been designed. With the development of research, more adenoviral particles are required. To meet the need of experimental and clinical applications, 293 cell lines which include adherent cell line and suspension cell line are frequently used in production. Methods used in production include batch, fed-batch and perfusion. Quality control of recombinant adenovirus vectors is also very important in the process of production. This review summarizes the production process of adenovirus vectors, mainly concentrated on the adenovirus infection mechanism and methods of production, purification and quantification.