The avian influenza virus (AIV) poses a significant threat to both the poultry industry and public health. Systematic identification of host factors involved in AIV infection in chicken is critical. In this study, we developed a comprehensive chicken genome-wide sgRNA library containing 76,350 sgRNAs, with 4-6 sgRNAs designed per gene. Then, we constructed a genome-wide CRISPR/Cas9 knockout chicken fibroblasts cells (DF-1 cells) library, covering 99.9 % of the total sgRNAs. Following multiple rounds of survival selection during AIV infection, 706 potential genes were identified, including 107 genes previously associated with AIV infection. These candidate genes were primarily involved in ubiquitin-related pathways, RNA transport, endocytosis, and other cellular processes. Among these, 18 novel hits were selected and confirmed to contribute to AIV-induced cell death, with eight genes specifically implicated in AIV proliferation. Notably, RNF2 was found to negatively regulate interferon-stimulated genes (ISGs), DCP1A was suggested to influence gene expression linked to AIV proliferation, and CREB3L3 may regulate membrane cholesterol levels during AIV invasion, further validating the screening results. This study identified 599 potential chicken genes involved in AIV infection, providing a foundation for a deeper understanding of the mechanisms underlying AIV infection in avian cells.