The evaluation of progesterone (P4) concentration is a valuable tool in assessing physiological reproductive events and reproductive disorders in bitches. A reliable and rapid (preferable, point of care) determination of P4 is advisable in most cases. Aims of this study were to evaluate a fluorescence enzyme immunoassay (FEIA) for canine serum P4 concentration by (i) the agreement with liquid chromatography–tandem mass spectrometry (LC/MS/MS), (ii) the association with vaginal cytology and (iii) the accuracy in the prediction of the parturition date calculated from the estimated day of ovulation. Serum samples were collected from client-owned bitches presented between 2011 and 2014 for the evaluation of their oestrous cycle, pregnancy or reproductive disorders. The agreement between FEIA and LC/MS/MS, evaluated on 19 samples, was statistically significant (R2 = 95.7%, p < 0.001), although FEIA showed significantly higher values than LC/MS/MS (p < 0.05). In the different phases of oestrous cycle, as determined by vaginal cytology, P4 concentrations (by FEIA) were statistically different (p < 0.05): anoestrus (n = 7) 0.38 ± 0.14 ng/ml, proestrus (n = 14) 1.04 ± 0.67 ng/ml and oestrus (n = 72) 6.8 ± 7.26 ng/ml. Mean pregnancy length from the estimated day of ovulation was 62.9 ± 1.8 days. In 13 of 22 (59.1%), 19 of 22 (86.3%) and 21 of 22 (95.5%) bitches pregnancy lasted 63 ± 1, 63 ± 2 and 63 ± 3 days, respectively. Three pregnancies were outside the 61–65 days range (60, 60 and 67 days). In conclusion, the FEIA method employed can be considered reliable and, in association with vaginal cytology, effective in evaluating the canine oestrous cycle.