Objectives The diagnosis of Non-Tuberculous Mycobacteria (NTM) infections is very challenging due to the polymorphic nature of these bacteria and to the pre-existing pathology in people with Cystic Fibrosis (pwCF). Recently, serological tests were developed for the detection of NTM, including M. avium complex and M. abscessus complex in pwCF, based on the detection of specific IgG and/or IgA against glycopeptidolipid, parietal extracts or virulence factors. Methods Sera from the CF patient population (French National Cohort CIMeNT, RCB ID N°: 2017-A00025-48) corresponding to 847 samples were probed by ELISA for the presence of IgG and IgA to establish or rule out prior a contact with NTM. Two previously reported M. abscessus antigenic preparations were assayed: the parietal extract purified for its TLR2-dependent proinflammatory activity (TLR2ef), and the virulence factor phospholipase C (PLC). Data from 4 different Ig/antigen combinations were analysed: IgG/TLR2ef, IgG/PLC, IgA/TLR-2ef and IgA/PLC. Results In this national prevalence survey, we included 856 CF patients among which 76 had one or more NTM positive sputum culture. Almost 78% of pwCF were negative for the four ELISA tests; 23% were positive for one, 11.4% positive for two, 5.7% positive for three and 3.2% positive for all four assays. Considering the positivity for at least two assays, the sensitivity was 69% and the specificity 81%. Knowing the prevalence of NTM positive sputum culture in this tested population, the positive and negative predictive values were 21% and 96%, respectively. Conclusions The very high negative predictive value suggests the usefulness of this seric assay to exclude a diagnosis of NTM infection. In the era of CFTR modulator therapy, associated with the decreasing number and volume of sputum samples, this method may prove useful as a first screening tool to rule out NTM infection in pwCF.