Serum carcinoembryonic antigen (CEA), cancer antigen 15-3 (CA 15-3), miR-21, and miR-29b levels are biomarkers that have been studied in human breast cancer (HBC).Given the similarity between canine mammary tumors (CMTs) and HBC, this study aimed to evaluate the potential of CEA, CA 15-3, miR-21, and miR-29b as diagnostic and prognostic biomarkers of CMTs.A total of 27 female dogs were divided into the non-tumor (n = 11), benign CMT (n = 8), and malignant CMT (n = 8) groups.Biomarker levels were compared among the groups.CEA levels were not significantly increased in the benign or malignant CMT group.CA 15-3 levels were higher in the malignant CMT group than in the benign CMT group, although the difference was not significant.Postoperative changes in CA 15-3 and CEA levels were not significantly correlated with histopathologic result and prognosis.miR-21 and miR-29b levels were not significantly different among the groups.Postoperative changes in miRNA levels were not significantly correlated with histopathologic result.miR-21 and miR-29b levels were increased postoperatively in patients with poor prognosis, with an increase in miR-29b levels significantly associated with poor prognosis.CEA has limited utility as both a diagnostic and prognostic biomarker in CMT.In contrast, CA 15-3 and miR-21 show promise as diagnostic and prognostic markers, respectively, and miR-29b demonstrates significant value as a prognostic biomarker.The elevated levels of CEA, CA 15-3, miR-21, and miR-29b observed in non-tumorous conditions indicate that they be used in conjunction with other indicators rather than as sole markers.