The present invention is related to a biomarker for diagnosis of canine cancer, wherein the biomarker is KMO (kynureinie 3-monooxygenase) gene, and the canine cancers including CTVT (canine transmissible venereal tumor) and MGT (mammary gland tumor). The expression level of KMO gene in canine malignant tumor tissues is up-regulated as compared with benign tissues. Furthermore, the expression level of KMO gene in malignant tumor tissue is higher than in the non-metastasis tumor tissue. By detection of the expression level of the present invention biomarker in suspecting tissue specimen, malignancy of tumor tissues can be determined correctly and rapidly.