RNA interference (RNAi) is a key antiviral immune mechanism in eukaryotes. However, antiviral RNAi in vertebrates has only been observed in cells with poor interferon systems or in viral suppressors of RNAi (VSR) deficiency virus infections. Our research discovered that infecting macrophages with wild-type coronavirus (Infectious bronchitis virus, IBV) and influenza viruses (Avian influenza virus, AIV) can trigger RNAi antiviral immunity and produce a certain amount of virus-derived siRNA (vsiRNA). These vsiRNAs have an inhibitory effect on the virus and carry out targeted silencing along the Dicer-Ago2-vsiRNA axis. Notably, these vsiRNAs are distributed throughout the virus's entire genome, with a predilection for A/U at the 5' and 3' termini of vsiRNA. In addition, Dicer cleavage produces vsiRNA based on the RWM motif, where R represents A/G, W represents A/C, and M represents A/U. We also discovered that avian LGP2 and MDA5 proteins positively impact the expression of the Dicer protein and the Dicer subtype "DicerM." Most importantly, the PS-vsiRNA plasmid combined with nanomaterial polyetherimide (PEI) showed excellent anti-virus activity in specific-pathogen-free (SPF) chickens. These findings show that RNA viruses trigger the production of the vsiRNA in avian somatic cells, which is of great significance for the application of therapeutic vaccines.