Background: Fraction of exhaled nitric oxide (FeNO) is used for diagnosing and monitoring asthma in children, but the influence of allergic sensitization is still poorly understood. Here, we investigate how asthma and allergic sensitization influence FeNO levels during childhood. Methods: We investigated the associations between asthma, aeroallergen sensitization, and FeNO measured from age 5–18 years in the COPSAC₂₀₀₀ birth cohort of 411 children using repeated measurement mixed models adjusted for gestational age, sex, concurrent airway infection, inhaled corticosteroids, and tobacco exposure. Replication was sought in the similarly designed COPSAC₂₀₁₀ cohort of 700 children. Results: In the COPSAC₂₀₀₀ cohort, 133 had asthma between age 5 and 18 years, and in the COPSAC₂₀₁₀ cohort, 112 had asthma between age 5 and 10 years. In the COPSAC₂₀₀₀ cohort, asthma and aeroallergen sensitization were both associated with higher FeNO from age 5 to 18 years: adjusted geometric mean ratio (aGMR), 1.22 (1.08–1.35), p <.01, and 1.41 (1.21–1.65), p < 0.001, respectively. However, asthma was associated with increased FeNO among children with aeroallergen sensitization: 1.44 (1.23–1.69), p <.0001, whereas asthma was associated with decreased FeNO among nonsensitized children: 0.80 (0.65–0.99), p =.05 (p-interaction<.0001 for asthma x sensitization). Replication in the COPSAC₂₀₁₀ cohort showed similar results (p-interaction <.01). Further, blood eosinophil count, total-IgE, bronchodilator response, and bronchial hyperreactivity were all associated with increased FeNO among children sensitized to aeroallergens, but not among nonsensitized children. Conclusion: Fraction of exhaled nitric oxide is elevated through childhood in children with asthma and is correlated with asthma-associated traits depending on the presence of aeroallergen sensitization. These findings indicate that FeNO is only a valid asthma biomarker in children with concurrent aeroallergen sensitization, which is important for guideline recommendations on the clinical use of FeNO.