H9N2 subtype avian influenza virus (AIV) remains a major threat to poultry industry. Our previously developed live-attenuated vaccine candidate rTX-NS1-128(mut) demonstrated promising immunogenicity, but its truncated NS1 gene reduced replication in MDCK cells relative to the parental rTX strain. In this study, we engineered an MDCK derived cell line (2G8D5) to enhance replication of interferon-sensitive AIV candidates. Using lentiviral transduction, 2G8D5 constitutively express the full-length NS1, thereby dampening type I interferon responses. rTX-NS1-128(mut) reached higher infectious titers in 2G8D5 than in standard MDCK cells. Over serial passages in 2G8D5, the virus remained genetically stable, with no HA gene mutations and no appreciable variation TCID?? and EID??. In specific-pathogen-free (SPF) chickens, virus produced in 2G8D5 elicited hemagglutination inhibition (HI) titers and IgY/IgA levels comparable to those induced by virus grown in SPF chicken embryo. Cytokine profiling showed similar IL-4, IL-5, and IFN-γ expression between groups. Upon challenge, vaccinated chickens exhibited reduced viral shedding; only two chickens per group shed virus at 3 days post-challenge (d.p.c.). In conclusion, these findings highlight the potential of the 2G8D5 as a robust platform for producing rTX-NS1-128(mut) and underscore its significance in the development of live attenuated vaccines against H9 subtype avian influenza virus.