Background Almost 30% of the premature infants have low body weight and bone mineral density due to prematurity. There is no consensus of screening premature neonates for metabolic bone disease; therefore, it is important to investigate the use of bone biochemical parameters. Latest studies involved the activity of acetylcholinesterase as a mediator in bone remodeling. It is hypothesized that there is a possible correlation of bone biochemical biomarkers and acetylcholinesterase (AChE) activity in premature infants. Methods We studied 50 neonates (26 preterm with gestational age <32 weeks, 24 full-term). Clinical data (sex, gestational week) and anthropometric parameters (body weight) were recorded. We directly measured the bone biochemical markers in serum such as alkaline phosphatase (ALP), calcium (Ca), phosphorus (P), magnesium (Mg) and parathyroid hormone (PTH). In addition, we measured the AChE activity. Results ALP and parathyroid hormone levels were higher, but Ca, P and AChE were lower in premature neonates group compared with full-term ones. There is a significant positive correlation of gestational age with body weight, Ca and AChE. A significant negative correlation was observed for ALP and PTH with gestational age. Conclusions We found a gestational age-related increase of AChE activity. There were significant relationships between AChE activity with P and PTH.