We investigated whether prepartum levels of serum bone biomarkers are related to the degree of parturient hypocalcaemia and risk of milk fever (MF) in dairy cows with advancing parity. A total of 58 late-pregnant cattle were assigned to four groups: nulliparous, primiparous, multiparous in the 2nd lactation and multiparous in the 3rd–5th lactation. The multiparous cows were further assigned to MF and non-MF groups according to the onset of MF. Serum samples were obtained from the cows during the 3 weeks prepartum to 5 d postpartum period for the measurement of serum calcium (Ca) and three bone biomarkers: tartrate-resistant acid phosphatase isoform 5b (TRAP5b), osteoprotegerin (OPG) and bone isoenzyme of alkaline phosphatase (ALP3). The ratios of OPG to TRAP5b (O/T ratio) and ALP3 to TRAP5b (A/T ratio) were calculated. The data from all cattle showed that the severity of hypocalcaemia at parturition increased with advancing parity/age. The MF cows had elevated serum TRAP5b activity and a decreased O/T ratio after parturition, suggesting an increased number of osteoclasts due to osteoclastogenesis, in response to severe hypocalcaemia. The MF cows showed lower serum ALP3 activity during the 3 weeks prepartum than the non-MF cows, therefore, prepartum osteoblast function was likely weak in the MF cows. During the 2–3 weeks prepartum, serum ALP3 activity and the A/T ratio had moderate associations with the serum Ca concentration at day 0 (day of calving) in the multiparous cows, and receiver operating characteristic curve analysis revealed that ALP3 activity had excellent ability to predict MF. In conclusion, prepartum serum ALP3 activity is a promising biomarker to predict MF in multiparous cows.