With the rapid development of gene therapy, gene-based medicine with adenovirus as vectors has become a new method for disease treatment. However, there are still enormous challenges in the large-scale production of adenoviruses for clinical use. Recent reports show that ion-exchange chromatography (IEC) is an effective tool for the isolation and purification of adenovirus. However, during the separation and purification, host cell protein and DNA, as well as serum from the culture medium, can non-specifically occupy numerous binding sites of the chromatography packings, thereby reducing the binding between the adenovirus and packing media. We here report a novel method for highly efficient purification of adenoviruses by increasing the salt concentrations of the samples to be ultrafiltrated by tangential flow filtration, the diafiltration buffer, and the samples for IEC purification. This method could significantly remove a large amount of serum proteins and host cell proteins, increase the amount of sample loaded on the IEC column, and improve the binding of the adenovirus samples to the packing media. A purity of > 95% could be obtained after one chromatography operation, and the number of purification steps and the amount of used packing media were reduced. The method is simple, economical, and efficient, and has excellent applications. Copyright © 2019 Elsevier B.V. All rights reserved.