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Canine mammary tumor is the most common tumor in intact female dogs and poses a growing health burden due to its high malignancy rate and increasing canine populations. However, research on sarcomatous subtypes has been hindered by a lack of representative cell lines. Here, we successfully established a novel canine mammary liposarcoma cell line, designated CMLPS-N1, which represents the first such model derived from a spontaneous tumor. This cell line has been stably maintained for over 80 passages and exhibits an abnormal karyotype, high proliferative and migratory capacity, and strong tumorigenicity in mouse xenografts. Molecular profiling confirmed a phenotype consistent with liposarcoma (MDM2+) and mesenchymal origin (Vimentin+/N-cadherin+), alongside high-risk markers (p53+/Ki67+/Notch1), and hormone receptor expression (ER/PR), while being negative for epithelial (PCK) and HER-2 markers. We used functional assays, including cell proliferation, colony formation, wound healing, and transwell invasion, to confirm its aggressive phenotype. Furthermore, cytotoxicity testing with four chemotherapy agents further supports its utility as a preclinical model for therapeutic screening and mechanistic research. The establishment of CMLPS-N1 enriches the canine mammary tumor cell line repository and provides a valuable experimental model for studying disease mechanisms, developing therapies, and facilitating translational applications.