Pediatric adrenocortical carcinoma (pACC) is a rare and aggressive malignancy of high occurrence in Southern Brazil. pACC is characterized by the usual overproduction of dehydroepiandrosterone sulfate (DHEAS), whose detection in serum or plasma can be effective to the early diagnosis of the disease. Therefore, the present paper reports, for the first time, the construction and application of a label-free impedimetric immunosensor to detect DHEAS, which was based on the modification of an oxidized glassy carbon electrode with arginine-functionalized gold nanoparticles (AuNPs-ARG) and anti-DHEA IgM antibodies (ox-GCE/AuNPs-ARG/IgM). AuNPs-ARG was synthesized by a green route, and characterized by UV-VIS spectroscopy, FTIR, TEM, DLS, and XRD. The construction of ox-GCE/AuNPs-ARG/IgM was optimized through factorial design and response surface methodology. Cyclic voltammetry and electrochemical impedance spectroscopy measurements were employed to characterize the optimized immunosensor. The DHEAS detection principle was based on the variation of charge transfer resistance (∆Rct) relative to the Fe(CN)64-/3- electrochemical probe after immunoassays in the presence of the biomarker. A linear relationship between ∆Rct and DHEAS concentration was verified in the range from 10.0 to 110.0 µg dL-1, with a LOD of 7.4 µg dL-1. Besides the good sensitivity, the immunosensor displayed accuracy, stability, and specificity to detect DHEAS. The promising analytical performance of ox-GCE/AuNPs-ARG/IgM was confirmed by quantifying DHEAS in real patient plasma samples, with results that were comparable to the reference chemiluminescence assay. Our results suggest that the presented immunosensor can find clinical applications in the early diagnosis of pACC and to monitor DHEAS levels in other adrenal pathologies.