Fibroblast growth factor-23 (FGF-23) is a phosphaturic hormone used to monitor chronic kidney disease (CKD) in humans. The aims of this study were (1) to determine the intra- and interassay precision of the FGF-23 concentrations in dogs as measured via the Kainos ELISA FGF-23 kit, (2) to calculate a reference interval, and (3) to assess the correlation of the FGF-23 concentration with the hematological and biochemical parameters. The coefficient of variation was below 15% for both the intra- and interassay precision, indicating good reproducibility. The reference interval ranged between 95.8 (90% confidence interval: 44.6; 139.2) and 695.1 pg/mL (598.7; 799.1) based on 136 clinically healthy dogs, classified as such according to the information of treating veterinarians as well as the unremarkable results of hematology and biochemistry. The FGF-23 concentration differed significantly between dogs aged <9 and ≥9 years ( = 0.045). Four groups of 10 dogs each were retrospectively formed based on the creatinine concentration classification according to the IRIS staging. Correlation was the strongest for the renal parameters. Statistically significant differences in the FGF-23 concentration were demonstrated between the study groups I and III ( 0.001), I and IV ( 0.001), and II and IV ( = 0.005). There was a trend for a rising FGF-23 concentration in older dogs. Due to the wide reference interval, diagnostic cut-offs and/or subject-based FGF-23 reference values in each dog are needed for monitoring and clinical interpretation.