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CMTs are biologically heterogeneous neoplasms for which reliable prognostic biomarkers remain limited. This study investigated the clinical significance of serum cancer antigen 15-3 (CA 15-3) and the immunohistochemical expression of Ki-67 and cyclooxygenase-2 (COX-2) in CMTs. Fifty-four female dogs with histologically confirmed CMTs and twelve healthy controls were prospectively evaluated. Serum CA 15-3 concentrations were measured before surgery and at 21 and 90 days post-mastectomy, while Ki-67 and COX-2 expression were assessed in tumor tissues. CA 15-3 was undetectable in controls and significantly higher in malignant than benign neoplasms (p < 0.05), with moderate correlations with clinical stage (s = 0.59), histological grade (s = 0.64), and the number of nodules (s = 0.42). Levels remained elevated in advanced stages despite surgery. ROC analysis identified a CA 15-3 cutoff of 3.01 IU/mL (AUC = 0.92) for discriminating malignant from benign tumors. Ki-67 correlated with histological grade (ρ = 0.41) but showed limited prognostic accuracy (AUC = 0.63). COX-2 expression lacked significant associations and showed poor discriminatory power (AUC = 0.44). Survival analyses did not identify significant differences among groups. Principal component analysis demonstrated clustering of aggressive CMTs according to biomarker profile. These findings suggest that serum CA 15-3 may represent a useful adjunct prognostic biomarker in canine mammary oncology, while the combination of serum and tissue biomarkers may improve prognostic stratification and may guide therapeutic decision-making in veterinary oncology. These findings support the growing role of biomarker panels in the clinical management of CMTs.