Myxomatous mitral valve disease (MMVD) is the most common acquired heart valve disease in dogs and it may contribute to cardiovascular-renal axis disorders (CvRD) in dogs. Sensitive and early biomarkers of renal involvement are needed. In this prospective and observational study, 84 dogs were enrolled (20 healthy dogs and 64 dogs with MMVD, categorized using the American College of Veterinary Internal Medicine guidelines. Serum and urinary parameters were analyzed, including tubular biomarkers expressed as creatinine-ratios: urinary alkaline phosphatase (uALPc), gamma-glutamyl transferase (uGGTc), N-acetyl-β-D-glucosaminidase (uNAGc) and cystatin C (uCystc). uALPc, uGGTc and uNAGc were higher in MMVD than in controls; uALPc and uGGTc were increased from stage B1, uNAGc was higher in stages with cardiomegaly (B2 and C+D), and uCystc increased mainly in clinical stages (C+D). Serum renal markers increased only in clinical stages. ROC analysis showed good discrimination for MMVD with uALPc (AUC 0.87) and uGGTc (0.86); for cardiomegaly with uALPc (0.77) and uNAGc (0.75); and for congestive heart failure with SDMA (0.85) and uCystc (0.75). No urinary biomarker was associated with daily furosemide dose. Urinary tubular biomarkers, particularly uALPc and uGGTc, detect early CvRD in dogs with MMVD and complement traditional serum markers.