Zika virus (ZIKV) has recently become a global health challenge due to its rapid geographical expansion, since it is associated with serious neurological anomalies such as Guillain-Barré syndrome and microcephaly. Currently, the techniques for ZIKV diagnosis require labor-intensive, expensive and lengthy tests using sophisticated equipment. Moreover, false-positive or false-negative results can occur. In the present work, a DNA biosensor to detect ZIKV in real human serum samples was developed using an oxidized glassy carbon electrode (ox-GCE) modified with silsesquioxane-functionalized gold nanoparticles (AuNPs-SiPy). This nanohybrid was characterized by UV-Vis, FTIR and Raman spectroscopies, DLS, and XRD. The conditions for the immobilization of a ZIKV ssDNA probe on the electrode surface (ox-GCE-[AuNPs-SiPy]) were optimized by univariate and multivariate analysis. The optimized biosensor was characterized by CV, EIS and AFM experiments. The ZIKV target recognition was based on the variation of the charge transfer resistance (ΔRct) of the redox marker ([Fe(CN)6]3-/4-) used and the roughness (Rq) of the electrode surface. The proposed biosensor presented a LOD of 0.82 pmol L-1, with a linear range of 1.0 x10-12 - 1.0 x10-6 mol L-1. Moreover, the reported device showed a suitable stability and satisfactory sensitivity and selectivity to quantify ZIKV in human serum samples, which suggests its promising clinical applications for the early diagnosis of ZIKV-associated pathologies.