tInfluenza reverse genetics plays vital roles in understanding influenza molecular characteristics and vac-cine development. However, current influenza reverse genetics heavily depends on restriction enzymeand ligation for gene cloning. The traditional cloning process of influenza eight fragments for virus rescu-ing generally requires considerable work. To simplify and increase the pace of gene cloning for influenzareverse genetics system, we developed a rapid restriction enzyme-free ExnaseTM II-based in vitro recom-bination approach for influenza gene cloning. We used this strategy rapidly and successfully to cloneinfluenza eight genes both from viruses PR8 and H9N2 for virus rescuing. Our data demonstrate thatthe strategy developed here can accelerate the process of influenza gene cloning into reverse geneticssystem, and shows high potential for applications in both influenza basic and applied research.