Seasonal changes in hematology and serum biochemistry results, described by separate reference intervals for different seasons, have been reported in many animals. We developed a novel method to investigate seasonal variation in values and a reference tool (the reference curve) based on sine wave functions that, for suitable variables, represents data more appropriately than a fixed reference interval. We applied these techniques to values observed in blood samples from 126 adult wild platypuses (Ornithorhynchus anatinus; 58 females and 68 males). Samples were collected under isoflurane anesthesia from animals captured in the Inglis Catchment in northwest Tasmania. In general, packed cell volume (PCV), red cell count (RCC), and hemoglobin (Hb) values appeared to be lower than those in two studies that previously reported platypus hematology reference intervals. This likely resulted from reduced stress-related splenic contraction or isoflurane-associated splenic sequestration of red blood cells in our study. Reference curves were described for five variables (PCV, RCC, Hb, albumin, and magnesium). We found evidence that this seasonal variation may result from metabolic changes associated with seasonal variations in environmental temperature. These observations suggest that it is important for researchers reporting platypus hematology and serum biochemistry to look for seasonal changes in their data to ensure it is appropriately interpreted.