An ultrasensitive label-free electrochemical immunosensor was developed for hepatitis C antibodies (anti-HCV). Worldwide, it is estimated 71 million people have HCV infection in a chronic stage that may lead to cirrose and cancer. To achieve HCV elimination, health programs should include screening testing based on anti-HCV detection allowing the early-stage treatment. The immunosensor was based on a graphene oxide-polypyrrole (PPy–GO) film one-step electropolymerized on the electrode surface. Ultrasensitive anti-HCV detection was ensured by HCV antigen conjugated to biotin that was immobilized in a great amount on streptavidin-coated nanostructured surface. Analytical responses were obtained by anodic peaks from the square wave voltammetry in the presence of ferrocyanide/ferricyanide as a redox probe. This immunosensor exhibited a linear range from 2 to 14 ng mL −1 of anti-HCV and a limit of detection in the clinical range (1.63 ng mL −1 ). Furthermore, the immunosensor presented an efficient performance for the determination of anti-HCV in spiked serum samples, becoming this developed nanosensor as potential tools for early HCV diagnosis and screening. Supplementary Information The online version contains supplementary material available at 10.1007/s10853-022-06992-5.