Numerous specific biomarkers with a prognostic and diagnostic value comparable to histopathological findings are now used for non-invasive diagnosis of prostate diseases in humans. Meanwhile, as far as dogs are concerned, the diagnosis of prostate disorders is based solely on clinical examination and ultrasound (USG). Therefore, the aim of the study was to assess the usefulness of two biomarkers, i.e. miRNA-129 and VEGF for the diagnosis of BPH in dogs. The study involved 40 dogs divided into three groups. Group I (n = 9) comprised healthy dogs up to the age of 5 years, Group II (n = 17) comprised dogs between the ages of 5–10 suffering from BPH as confirmed by the examination and Group III (n = 14) comprising dogs over 10 years of age, which also had BPH confirmed. The results demonstrated that dogs in group II and III exhibited a significant decrease in miRNA expression (P < 0.0001) and a significant increase in serum VEGF levels (P = 0.025) when compared to the dogs in group I. There was also a positive correlation between the prostate size and VEGF level. The findings led to the conclusion that the determination of miRNA-129 and VEGF can significantly contribute to the diagnosis of prostate disorders in dogs.