Calcitonin (CT) assay is of considerable importance in the routine evaluation of thyroid nodules and for screening and follow-up of patients with medullary thyroid carcinoma and their relatives. Aim of this study was to assess the reference ranges for CT levels in healthy children and to evaluate possible differences in CT levels between sex and age. Serum CT levels were measured by a commercially available two-site chemiluminescence immunometric assay (sensitivity = 0.2 pg/ml). The ILMA recognizes the mature monomeric form of CT. We evaluated a cohort of 125 healthy children and compared these results with those from 98 healthy adult men and women. The ranges for human CT in children were <0.2–11.7 pg/ml and <0.2–17 pg/ml for female and male, respectively. No gender differences were observed in children population, though higher CT levels were observed in males. Serum CT levels did not correlate with age. Adult female had statistically significant lower CT levels than female children (p ≤0.05). In the adult population, males showed levels of CT higher than females. In conclusion, this study provide normal range for children population at different age with a sensible two-site chemiluminescent immunoassay. Since the normal range of serum CT levels is wider in healthy children than in adults, these data suggest establishing different normal range values in different age groups.