The measurement of biochemical markers of bone turnover is essential in the study of skeletal metabolism in health and diseases. Due to variations in the rate of bone growth in different age groups and possible ethnic differences, age-specific reference ranges for biochemical markers should be established in a particular pediatric population. In this study, biochemical markers of bone formation, bone-specific alkaline phosphatase (BAP), and osteocalcin (OC) in healthy Thai children and adolescents aged 9 to 18 years were evaluated in relation to their ages and pubertal development. Serum BAP levels in boys increased with age and peaked at about 12 to 13 years. In contrast, there was a progressive decline of serum BAP levels with advancing age in girls older than 9 years. Serum OC also increased with age and reached a peak at ages 12 and 13 years in girls and boys, respectively. In addition, both serum BAP and OC levels also varied with pubertal stages. The BAP levels in boys increased sharply at pubertal stage 3 and decreased at pubertal stage 5. In girls, the BAP levels showed a fairly constant high level up to stage 3, followed by a remarkable decrease thereafter. The OC levels in boys increased sharply at pubertal stage 4 and decreased thereafter. In girls, OC started to increase at pubertal stage 3 with no subsequent changes. The levels of serum BAP and OC were higher in boys than in girls at pubertal stages 3 to 5 and at stages 2, 4, and 5, respectively. Moreover, only serum BAP level showed significant positive correlation with height velocity in both genders. In multiple regression analyses, gender, age, and pubertal stage were consistently correlated with both serum BAP and OC levels. In summary, male and female adolescents have different patterns of changes in biochemical markers of bone formation.