Aim: This study, the cut-off point of biochemical parameters in bitches with mammary tumors was determined and its usability as a biomarker for diagnosis was investigated. Materials and Methods: Bitches that presented to our clinic for routine ovariohysterectomy (control group, n = 14) and suffer from mammary tumors (CMT group, n = 14) were included in the study. Biochemical parameters including albümin (ALB), alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), gammaglutamyltransferase (GGT), total protein (TP), direct bilirubin (DB), total bilirubin (TP), creatine, creatine kinase-N-acetyl-cysteine (CK-NAC), urea, phosphorus (P), magnesium (Mg), and calcium (Ca) concentrations were measured with an automated chemistry analyzer. Results: The ALP (p: 0.048), ALT (p < 0.000), AST (p: 0.043), GGT (p: 0.013), TP (p: 0.010), DB (p: 0.043), TP (p: 0.018), creatine (p < 0.000), P (p < 0.000), and CK-NAC (p < 0.000) concentrations were found to be higher in the CMT group than in the control group. Although Mg (p < 0.000) concentration was higher in the control group, there was no statistical significance for ALB, urea, and Ca concentrations among the groups (p > 0.05). It was seen that ALP, ALT, AST, GGT, TP, and creatine concentrations could be used to diagnose mammary tumors, with area under the curve (AUC) values of 0.656, 0.831, 0.610, 0.792, 0.942, and 0.974, respectively. Conclusion: It was concluded that ALP, ALT, AST, GGT, TP, and creatine could be helpful diagnostic tools for canine mammary tumors.