Yellow fever is an arboviral disease that causes thousands of deaths every year in Africa and the Americas. However， few commercial diagnostic kits are available. Non-structural protein 1 (NS1) is an early marker of several flavivirus infections and is widely used to diagnose dengue virus (DENV) infection. Nonetheless， little is known about the dynamics of Yellow fever virus (YFV) NS1 expression and secretion， to encourage its use in diagnosis. To tackle this issue， we developed a quantitative NS1-capture ELISA specific for YFV using a monoclonal antibody and recombinant NS1 protein. This test was used to quantify NS1 in mosquito and human cell line cultures infected with vaccine and wild YFV strains. Our results showed that NS1 was detectable in the culture supernatants of both cell lines; however， a higher concentration was maintained as cell-associated rather than secreted into the extracellular milieu. A panel of 73 human samples was used to demonstrate the suitability of YFV NS1 as a diagnostic tool， resulting in 80% sensitivity， 100% specificity， a 100% positive predictive value and a 95.5% negative predictive value compared with RT-PCR. Overall， the developed NS1-capture ELISA showed potential as a promising assay for the detection of early YF infection.