Background: The role of immunoglobulin E (IgE) –dependent mechanisms leading to exacerbations and respiratory function worsening in cystic fibrosis patients is well acknowledged but not easy to characterize. We hypothesize that the availability of new tools such as allergen microarrays may contribute to a better understanding of disease-related local lung IgE responses. Method: We developed a three-step protocol to characterize specific IgE (sIgE) from cystic fibrosis patients’ sputum. First, sputum was fluxed and filtered before an overnight freeze-drying step. After re-suspending, we performed an allergen microarray with more than 300 allergenic extracts and components. Results: The protocol was applied to sputum samples from 22 cystic fibrosis patients, including 5 lung transplanted patients. The age was similar between non-transplanted and lung transplanted patients, with a median of 29 (IQR 17–72) and 39 (18–53) years, respectively. Non-transplanted patients had a higher overall IgE response against environmental allergens, especially animal dander allergens (p = 0.0004) and house dust mites (P = 0.03). IgE responses against molds did not differ between groups. No correlation was found between each patient’s IgE response to fungal extracts and components and fungal colonization. However, presence of sputum sIgE against fungal allergens, notably Cladosporium herbarum, was correlated with ongoing exacerbation. Conclusion: We have developed a technically simple protocol allowing non-invasive investigation of an extended analysis of local humoral immunity in cystic fibrosis patients. Exploration of sputum through allergen microarray may guide the identification of culprit agents leading to exacerbations and contribute to better management of cystic fibrosis patients.