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Background: The clinical application of canine mesenchymal stem cells (MSCs) necessitates efficient and safe culture methods to produce large quantities of cells. Traditionally, fetal bovine serum (FBS) has been used for MSC expansion, but it carries risks such as contamination and adverse immune responses. Methods: In this study, we investigate the efficacy and efficiency of canine allogeneic serum as an effective alternative to FBS for the in vitro culture of canine MSCs. We measured the population doubling time of canine MSCs in allogeneic serum conditions and utilized qRT-PCR, flowcytometric analysis, and cellular staining/color-metric assay for investigating its effects on cellular senescence during long-term culture and the expression of key pluripotency-related transcriptomes. Results: Our findings demonstrate that canine MSCs cultured with allogeneic serum exhibited enhanced proliferation rates, reduced cellular senescence, and lower apoptosis levels compared to those cultured with FBS. Additionally, the expression of key pluripotency-related transcription factors, including Oct4, Sox2, and Nanog, was increased in canine MSCs cultured with allogeneic serum. Conclusions: These results highlight the potential of canine allogeneic serum to provide a safer and more effective culture environment, supporting the large-scale expansion and maintenance of canine MSCs for clinical applications.