Recombinant adenoviruses have broad applications for gene delivery and expression. Furthermore, the adenovirus packaging system facilitates the expression of RNA-guided CRISPR/Cas9 nuclease complexes. In this study, we developed a novel system, named AdBlue, for the construction of recombinant adenoviruses using an enzymatic assembly strategy. This system could significantly reduce the time and labor required to generate adenoviral vectors. When applied to CRISPR/Cas9 design, it simplifies the preparation of recombinant adenoviruses carrying nuclease complexes and can induce high levels of site-specific mutagenesis. Our system has outstanding advantages for adenovirus preparation and could be a useful molecular engineering tool for gene delivery and editing.