In this work, an electrochemical sensor was created based on pencil graphite electrode (PGE) modified by copper nanoparticles (CuNPs) and poly- neutral red (poly-NR) for folic acid (FA) analysis. The morphological properties of the structured sensor (CuNPs/poly-NR/PGE) were characterized by an electrochemical impedance spectroscopy (EIS), scanning electron microscopy (SEM), and energy-dispersive X-ray spectroscopy (EDS). Subsequently, various parameters which affect the electrochemical performance of the modified electrode toward folic acid analysis such as the pH of buffer solution, type and concentration of buffer solution, and amount of modifiers on the electrode surface were discussed. The modified electrode showed excellent electrocatalytic ability toward FA oxidation with an irreversible behavior in pH 11 Britton-Rabinson (BR) buffer solution. Square wave voltammetry (SWV) was used toward FA determination. A calibration graph was plotted in optimized conditions, by the alteration of anodic peak currents as a function of FA concentration in the linear range of 1-1000 μM with a detection limit of 300 nM (S/N = 3). The CuNPs/poly-NR/PGE was applied for the determination of FA in blood serum sample as real sample and the good accuracy of the sensor that was corroborated by admissible betterment values.