IntroductionEosinophil‐derived neurotoxin (EDN) is related to childhood asthma, while normal values are lacking. We aimed to document serum EDN levels at 1 and 3 years in general and in non‐atopic children, and explore if EDN levels differed by sex or were associated with preschool asthma at 3 years.MethodsFrom the PreventADALL birth cohort, we included 1233 children with EDN analysed using ImmunoCAP at 1 and/or 3 years. Non‐atopic children had no history of wheeze, asthma, allergic sensitization or atopic dermatitis. Preschool asthma was defined as having ≥3 episodes of bronchial obstruction between 2 and 3 years, plus doctor diagnosed asthma and/or asthma medication use by 3 years. The upper limit of normal (ULN) of EDN was defined as the 95th percentile. With Youden Index we calculated EDN cut‐off levels for risk of preschool asthma.ResultsThe overall median (ULN) EDN levels were 27.4 (121) μg/L at 1 year (n = 787), and 20.1 (87.8) μg/L at 3 years (n = 857). Non‐atopic children had EDN levels of 24.0 (107) μg/L at 1 year (n = 147), and 17.3 (84.6) μg/L at 3 years (n = 173). EDN levels were higher in boys compared to girls; 32.0 (133) versus 24.5 (97.0) μg/L at 1 year, and 20.9 (96.3) versus 19.0 (72.4) μg/L at 3 years. Preschool asthma was observed in 109/892 (12.2%) children. Higher EDN levels at 1 (>26.7 μg/L) and 3 (≥20.5 μg/L) years were associated with preschool asthma; adjusted OR (95% CI) 2.20 (1.09, 4.41) and 4.68 (2.29, 9.55), respectively.Conclusion and Clinical RelevanceWe report EDN values in early childhood, demonstrating higher levels at 1 compared to 3 years and in boys compared to girls at both ages. Higher EDN levels at both ages were associated with preschool asthma. However, EDN cut‐off levels for preschool asthma were overall lower than the ULN of non‐atopic children, limiting translation into clinical practice.