This study presents the development of an electrochemical sensor based on a carbon paste electrode modified with nickel-cobalt multilayer nanowires (Co-Ni(MLNW)/CPE) for the detection of valganciclovir, an antiviral drug. The sensor was fabricated using an electrochemical deposition method, and its electrochemical behavior was investigated through cyclic voltammetry (CV) and differential pulse voltammetry (DPV). The influence of pH on the sensor's performance was extensively studied, revealing that the redox reaction of valganciclovir (VGCV) involves proton exchange, making pH optimization crucial. The results demonstrated that the sensor exhibited a wide linear range from 0.1 to 2000 nM, with a low detection limit of 0.03 nM at pH 7, the optimal condition for VGCV detection. Additionally, the sensor showed excellent stability, reproducibility, and selectivity, with negligible interference from common ions and biological molecules. The sensor's applicability was further validated through the determination of VGCV in human plasma samples, achieving a high recovery rate of 97.9%. These findings indicate that the proposed Co-Ni(MLNW)/CPE sensor is a promising tool for the accurate, sensitive, and reliable determination of VGCV in clinical and pharmaceutical settings.