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Dengue is an infectious viral disease transmitted by the Aedes aegypti mosquito, the control of which is complex. In addition, the clinical diagnosis is difficult to perform since it resembles other febrile infections; thus, the development of more effective methods to detect dengue virus (DV) has drawn increasing attention. The present study aimed to develop an impedimetric immunosensor for dengue diagnosis using a screen-printed electrode (SPE) functionalized with polymer films derived from 4-aminophenylacetic acid (4-APA). Data obtained from scanning electron microscopy (SEM) showed the deposition of a uniformly distributed material over the electrode surface. The immunosensor was based on the specific interaction between dengue antigen, NS1 protein, and anti-NS1 antibodies, IgG and IgM. In a characterization study using cyclic voltammetry (CV), the polymer film showed two oxidation peaks at +0.17 and + 0.35 V in 0.50 M sulfuric acid solution, indicating its adsorption and electroactivity at the SPE surface. Electrochemical impedance spectroscopy (EIS) measurements showed a higher charge transfer resistance (R) to the polymer film-modified SPE as compared with the bare SPE, corroborating a previous study. The best rNS1 concentration for immobilization was 1.00 ng/mL, and the immunoreaction time between the antigen (Ag) and the antibody (Ab) was 20 min. Dilutions of positive and negative clinical serum samples were evaluated by EIE, from which it was possible to elucidate, for the positive serum, that the more diluted the serum the greater the R. Negative serum also showed an analytical signal, probably due to the presence of non-specific antibodies; however, the generated signal presented values closer to the rNS1 signal, indicating good selectivity of the proposed platform. The experiments were repeated using bare SPE to verify the importance of the polymer film in biosensor construction. No significant difference was observed between these results. Graphical abstract Proposed schematic for the genosensor development.