Dendritic cells (DCs) are the most potent antigen-presenting cells and are the key link between the innate and adaptive immune response. Only a few reports with study populations of up to 50 individuals have been published with age-based reference values for DC subpopulations in healthy children. Therefore, we aimed to establish reference ranges in a larger study population of 100 healthy children, which allowed age-matched subgroups. Most previous studies were performed using a dual-platform approach. In this study, a single-platform approach in a lyse no-wash procedure was used. DC subpopulations were defined as follows: CD45(+) CD85k(+) HLA-DR(+) CD14(-) CD16(-) CD33(+) cells as myeloid DCs (mDCs) and CD45(+) CD85k(+) HLA-DR(+) CD14(-) CD16(-) CD123(+) cells as plasmacytoid DCs (pDCs). Reference ranges were established using a semi-parametric regression of age-matched absolute and relative DC counts. We found a significant decline with increasing age in the medians of mDCs (P = 0.0003) and pDCs per μl peripheral blood (PB) (P = 0.004) and in the 50%, 90% and 95% reference ranges. We also identified significantly lower absolute cell counts of mDCs per μl PB in girls than in boys for all age groups (P = 0.0015). Due to the larger paediatric study population and single-platform approach, this study may give a more precise overview of the normal age-matched development of DC subpopulations and may provide a basis for analyzing abnormal DC counts in different illnesses or therapies such as post stem cell transplantation.