Since MDCK cells are inherently tumorigenic, their safety in vaccine production has long been a concern; thus, establishing a screening method for low-tumorigenic cells is of great significance for influenza vaccine development. This study successfully obtained a low-tumorigenic MDCK cell line through monoclonal screening and systematically evaluated its potential as a cellular substrate for influenza vaccines using male nude mice (BALB/c nu/nu, 4-7 weeks old) for tumorigenicity assessment. Comprehensive analysis of the biological characteristics of the screened cells-including growth curves and transcriptomic features-showed that the cell line exhibits stable growth and consistent traits. Transcriptomic comparison was performed between two defined biological states: parental MDCK cells (SQ group) and the low-tumorigenic clone MDCK-20B9 (SH group). Transcriptomic analysis revealed good dispersion among samples and an overall consistent gene expression distribution. Differential expression analysis identified a total of 2198 differentially expressed genes, including 902 upregulated and 1296 downregulated genes. GO functional enrichment analysis indicated that these genes are mainly involved in biological processes such as acute-phase response, retinol metabolism, mitotic chromosome condensation, and cell migration; are enriched in cellular components such as kinetochores and the extracellular matrix; and are associated with molecular functions including calcium ion binding and the Wnt signaling pathway. KEGG pathway analysis further revealed that the differentially expressed genes are significantly enriched in key pathways such as cancer pathways, cell cycle, and cell adhesion molecules. The expression trends of five key differentially expressed genes were validated by RT-qPCR. In summary, this study successfully screened a stable and consistent low-tumorigenic MDCK cell line, providing a theoretical basis and practical foundation for its use as a cellular substrate in influenza vaccine development.