In the present paper, electrochemical quantification of an opioid analgesic butorphanol (BTR) at copper oxide/polyaniline nanocomposite modified glassy carbon electrode (CuO/PANI/GCE) has been discussed. The electroactive surface was produced by drop casting a suspension of CuO/PANI onto the surface of glassy carbon electrode. The nanocomposite was characterized by XRD, XPS, EDX, TEM, UV–Vis, FT-IR, Raman and FESEM techniques. Electrochemical impedance spectroscopy (EIS) technique was utilized to study the enhanced charge transfer phenomenon occurring at the surface of the modified sensor. The two electron oxidation process of BTR at CuO/PANI/GCE was found to be a function of pH (5.6) of the medium, scan rate and concentration of BTR. The optimized voltammetric response is observed in Britton-Robinson buffer, which shows an occurrence of an irreversible diffusion-controlled phenomenon where the anodic peak current is proportional to the BTR concentration within the range of 1.99 to 9.95 µg/L, with a detection limit of 1.38 µg/L (LOD) and the quantification limit of 4.18 µg/L (LOQ). The applicability of the proposed method was also studied for the determination of BTR in pharmaceutical formulation and human blood serum.