The excessive intake of antioxidant drugs due to veterinary and human medical treatment poses serious health risks to both humans and animals. In this context, the monitoring of antioxidant drugs in animal and human biological systems is important and has provoked a universal scientific challenge. Therefore, it is of the utmost importance to develop cost-effective electrochemical sensing electrode materials for the sensing of bio-analytes and pharmaceutical drugs in biological samples. In this work, electro-etched carbon nanofibers (CNFs) were coated with bottlebrush-like zinc oxide (ZnO) nanowires via a facile hydrothermal method for the preparation of a CNF-ZnO nanocomposite as a promising electrode material for the trace level detection of silymarin. The fabricated CNF-ZnO glassy carbon electrode (GCE) demonstrated a higher anodic peak current, lower peak potential, and excellent sensing performance of silymarin compared to GCEs fabricated with ZnO or CNFs. The CNF-ZnO-GCE exhibited an appreciable linear response range (2–123 nM), good sensitivity (1.728 μA∙nM⁻¹ cm²), and an ultra-low detection limit (1 nM). We also successfully showcased its specific sensing behavior for the sensing of silymarin in the presence of structurally similar compounds and metal ions. Furthermore, the CNF-ZnO-GCE was effectively applied to real biological and pharmacological samples for the efficient determination of silymarin, with which we obtained satisfactory recovery values.