Background: For optimal medical decision-making, harmonized reference intervals for estradiol for different ages and both sexes are needed. Our aim was to establish reference intervals using a highly accurate and traceable LC-MS/MS method and to compare these with reference intervals in literature. Methods: Estradiol was measured in serum obtained daily during the menstrual cycle of 30 healthy premenopausal women and in serum of 64 men and 33 postmenopausal women. The accuracy of our LC-MS/MS method was demonstrated by a method comparison with the CDC reference method. Results: Our LC-MS/MS method was traceable to the reference method. Estradiol reference interval during the early follicular phase (days −15 to −6) was 31–771 pmol/L; during the late follicular phase (days −5 to −1) 104–1742 pmol/L; during the LH peak (day 0) 275–2864 pmol/L; during the early luteal phase (days +1 to +4) 95–1188 pmol/L; during mid luteal phase (days +5 to +9) 151–1941 pmol/L; during late luteal phase (days +10 to +14) 39–1769 pmol/L. The reference interval for men was 12–136 pmol/L and for postmenopausal women <26 pmol/L. Conclusions: The established estradiol reference intervals can be used for all traceable LC-MS/MS methods for medical-decision making.