Temodal is an anticancer drug with many side effects such as loss of appetite and fatigue. Given that the determination of Temodal in patients treated with is a necessity, this research was focused on developing a DNA-based sensor for the determination of Temodal. For this purpose, the pencil graphite electrode was modified with Au nanoparticle and ds-DNA (ds-DNA/Au-NPs/PGE). The resulting electrode (ds-DNA/Au-NPs/PGE) was found to produce a linear response to concentration Temodal in the range of 5.0 nM-45.0 mu M with a detection limit of 1.0 nM. The response was attributed to the intercalation of Temodal with guanine species of the DNA species. The presence of Au nanoparticle at the surface of the modified PGE increases the active surface area to over 5.1-times and hence helps improve the detection limit of the voltammetric analysis. The factors affecting the performance of the biosensor (e.g. DNA concentration, ds-DNA/Temodal interaction time, and temperature) were optimized. A docking investigation was further used to evaluate the intercalation between guanine base with Temodal and the minor groove interaction of guanine bases of the ds-DNA with Temodal. The theoretical investigation confirmed that Temodal is stabilized by the intercalating site of DNA through pi-pi interactions and two hydrogen bonds with nucleotides and the binding energy was determined to be -5.75 Kcal/mol.