In this study, a new voltammetric nanosensor was constructed by the decoration of acetylene black (AB) with gold nanoparticles (AuNPs) using glassy carbon paste (GCP) as a cross linker (nano-AB-AuNPs/GCPE) for determination of anticancer topotecan (TPT) drug. Moreover, from the response characteristics of cyclic voltammetry (CV) and square wave voltammetry (SWV), it was observed that TPT could be effectively accumulated at nano-AB-AuNPs/GCPE and resulted in a sensitive anodic peak current in PBS of pH 6.0. The surface area, electrical conductivity and catalytic performance of the modified electrode improved on account of the formation of nano-AB-AuNPs materials, thereby enhancing the sensitivity of the nanosensor. Under optimal conditions, a novel electrochemical method based on the nano-AB-AuNPs/GCPE sensor with high selectivity, high sensitivity and high anti-interference ability was developed for the determination of TPT. Furthermore, the electrochemical oxidation of TPT reveals an excellent linearity in the range of 1.99 x 10(-9) to 6.71 x 10(-7) M of TPT with a detection limit of 1.64 x 10(-11) M (6.91 x 10(-3) ng/mL). The TPT as a preconcentration agent showed chemical selectivity, so, the nanosensor was successfully applied for the determination of TPT in the presence of high concentrations of uric acid. Moreover, the method was employed efficiently to detect TPT in blood serum and urine samples, with satisfactory recoveries ranging from 98.16% to 100.76%.