Pyridoxine is analyzed using square wave voltammetry (SWV) at copper nanoparticles (nano-Cu) modified poly-crystalline gold electrode (nano-Cu/Au). Nano-Cu/Au is fabricated by a potential scan electrodeosition technique. Nano-Cu/Au electrode has been characterized morphologically and electrochemically. The analysis of pyridoxine at nano-Cu/Au electrode is achieved utilizing the quenching of copper voltammetric response due to the complexation with pyridoxine forming an electroinactive complex. Pyridoxine selectively forms complex with copper ions (modifier), but not with Au (underlying substrate) as supported by UV/Vis spectrophotometry. Using SWV the calibration curve for pyridoxine analysis was obtained in the concentration range of 0.3–2.7 mM with high correlation coefficient. The proposed method has been successfully applied for the determination of pyridoxine in two dosage forms.