Viral infectious diseases seriously threaten human health. At present, the prevention and treatment of viral diseases depends primarily on vaccines and drugs. Commonly used research method include animal models or simple traditional two-dimensional (2D) isolation culture, but 2D cell behaviour is different from the human physiological microenvironment. Therefore, a new model that can simulate the human microenvironment is needed. Here, the nanoself-assembled peptide KLD-12 was used as a culture scaffold for 293T cells in threedimensional (3D) adenovirus culture. As a new 3D virus culture model, it simulates in vivo virus infection, and the model can produce infectious particles. In addition, the antiviral drug sensitivity between the 3D and 2D cultures was significantly different. We established a 3D adenovirus culture model that can be used for adenovirus proliferation and antiviral drug screening, as well as for gene therapy, vaccine research and other research. Additionally, it can partially replace animal models.