The incidence of infection by the dengue virus (DENV) has grown dramatically, reaching 128 countries in tropical and subtropical regions worldwide, with a pattern of hyper-endemicity. DENV is a mosquito-borne disease having four serotypes, one or two circulating in epidemic outbreaks. The diagnosis of DENV is challenging mainly due to the circulation of new viruses with remarkable similarities, such as Zika (ZIKV) that may cause fetal microcephaly. DENV affects 390 million people per year, but these numbers may be higher due to the underreported and misclassified cases. Recently, the NS1 nonstructural protein has been described in serum and urine of DENV and ZIKV patients, suggesting its use as a biomarker for screening since a negative NS1 sample confirms the absence of these infections. Herein, a label-free immunosensor comprising an assembled nanostructured thin film of carbon nanotube-ethylenediamine is described. The advantage of in situ electrosynthesis of polymer film is to allow major control of thickness and conductivity, in addition to designing the reactive groups for functionalization. A quartz crystal microbalance system was used to estimate the thickness of the polymeric film obtained. The anti-NS1 monoclonal antibodies were immobilized to carbon nanotubes by covalent linkage, permitting a high stability during measurements. Analytical responses to NS1 were obtained by differential pulse voltammetry (DPV), showing a linear range from 20 to 800 ng mL and reproducibility of 3.0%, with a limit of detection (LOD) of 6.8 ng mL. This immunosensor was capable of detecting ZIKV and DENV NS1 in spiked urine and real serum in a clinical range.Graphical abstract.