In this study, a new biosensor for amisulpride (ASP) determination is defined. The interaction between ASP and fish sperm double-stranded DNA (FSdsDNA) was examined by differential pulse voltammetry (DPV) method utilizing FSdsDNA modified pencil graphite electrode (PGE). Sensitive and reliable ASP determination and detection was performed in pH 4.80 acetate buffer (20% ethanol) before and after the interaction of ASP and FSdsDNA, acting by the change in guanine oxidation signals from the electro-active bases of FSdsDNA. In addition, analytical method validity tests were examined in this study. This modified electrode have used in 1–10 μg/mL determination interval for the quantitative determination of ASP in pharmaceutical forms and the limit of detection (LOD) has been found as 0.46 μg/mL. Also, the binding constants (Kb) of the ASP with FSdsDNA has also been calculated both UV-Vis and DPV techniques.