Accurately monitoring the blood levels of testosterone (TST) in a sensitive, simple, and rapid manner is of paramount importance for the diagnosis and treatment of various medical conditions, as well as for controlling pharmaceutical quality and facilitating doping detection. TST, the primary male sex hormone, plays a crucial role in facilitating human physical performance, protein synthesis, and the development of muscle mass. Consequently, TST and its analogues are frequently abused by athletes as performance-enhancing steroid in order to increase muscle mass and enhance their performance. The use of such steroids is strictly prohibited to guarantee fair play. In this study, we employed a pencil graphite electrode that was electrochemically modified with CuO nanoparticles (CuONPs) for determination of TST. The electrode response was significantly enhanced by approximately fourfold compared to the unmodified pencil graphite electrode (PGE) when electrodeposition CuONPs onto PGE surface was performed at a potential of −0.6 V for 200 s. The success of modification was confirmed through morphological analysis using scanning electron microscopy and energy-dispersive x-ray spectroscopy. Using square wave adsorptive stripping voltammetry analysis in Britton–Robinson buffer at pH 6.0, we demonstrated that the proposed sensor exhibited sensitivity to detect TST within a linear range of 5–200 nM. The detection limit of sensor was calculated 4.6 nM (1.32 ng ml−1). The sensor platform developed for the accurate, sensitive, and specific determination of TST holds tremendous potential for the development of point-of-care devices and their integration into lab-on-a-chip research.