BackgroundBenign prostatic hyperplasia (BPH) is the most common canine prostatic disorder. Although most or even all intact male dogs may develop BPH by 5–8 years of age, many show no clinical signs. Taking into account the non-specific character of clinical and ultrasonographic findings, a new diagnostic approach has recently been proposed based on the augmentation of blood canine prostate-specific arginine esterase (CPSE) in hyperplasic dogs. The aim of the present study was to verify CPSE levels in negative controls and hyperplasic dogs, considering cytological findings as the reference method and taking into account the fact that controls were middle-aged intact dogs (median of 5.0 years), contrarily to previous studies carried out with very young control dogs.ResultsSignificant differences of median CPSE levels were found between controls and hyperplasic dogs (29.1 versus 160.7 ng/mL, respectively); and significant positive correlations were found between median CPSE levels and age or prostatic volume (r = 0.549 and 0.448, respectively; p < 0.001). Sensitivity, specificity, positive and negative likelihood ratios put into evidence the good performance of the test. The agreement between methods was found to be very high, notably between CPSE levels and cytological results (Cohen’s kappa coefficients above 0.8).ConclusionsConsidering the results all together, measurement of CPSE is confirmed as a useful and accurate method and should be considered as an alternative or complementary tool to conventional methods for the diagnosis of BPH in middle-aged dogs.Electronic supplementary materialThe online version of this article (doi:10.1186/s12917-017-0996-5) contains supplementary material, which is available to authorized users.