Summary: Bone formation markers bone-specific alkaline phosphatase and osteocalcin are used in many clinical situations. Therefore, we calculated reference intervals for the two markers and investigated how they are influenced by several factors including sex and age. Furthermore, we established clinically relevant reference intervals for the two markers. Objective: The bone turnover markers (BTMs), bone-specific alkaline phosphatase (bone ALP), and osteocalcin (OC), are frequently measured formation markers. The purpose of this study was to establish reference intervals (RIs) for the two BTMs in a general adult Danish population. Methods: Bone ALP and OC were measured on the iSYS (IDS Plc) automatic analyzer in samples from the Danish Health2006 5-year follow-up study on serum from 2308 participants (54% women, age range 24–76). Participants with self-reported diagnosis of osteoporosis or receiving hormonal replacement were excluded from analyses while participants on hormonal contraceptives were included. Results: The geometric mean and 95%RI for bone ALP were 13.9 μg/L (7.6–25.6) for men and 13.8 μg/L (7.0–27.4) for women, while for OC 16.0 μg/L (7.5–34.4) for men and 18.6 μg/L (8.1–42.9) for women. Levels of bone ALP increased with increasing age (β 1.004, p < 0.001), while female sex had no effect. OC levels decreased with increasing age (β 0.998, p = 0.009) and increased with female sex (β 1.104, p < 0.001). Based on our findings, we propose for bone ALP and OC three clinical RIs for men based on age and three clinical RI for women based on age and menopausal status. Conclusion: The RI for bone ALP and OC varies with age and sex and the BTMs are influenced differently by the two factors. Consequently, the need for establishing valid RIs is of great importance before the full potential of BTM can be used in clinical practice.