Copper (II) oxide nanoparticles (CuONPs) were green synthesized using Ocimum basilicum leaves aqueous extract in which polyphenols act as reducing and stabilizing agents. The synthesized CuONPs were characterized using X-ray diffraction spectroscopy, Fourier transform infrared spectroscopy, transmission electron microscopy, selected area electron diffraction, and Brunauer–Emmett–Teller (BET) surface area analysis. The analyses indicated the formation of crystalline rod-like monoclinic pure CuONPs with a mean grain size of 15 nm, a surface area of 396 m2 g−1, and a total pore volume of 0.71 cm3 g−1. A glassy carbon electrode (GCE) was modified using the synthesized CuONPs and electropolymerized poly(methyl orange) (PMO). The modified PMO/CuONPs/GCE electrode was electrochemically characterized and applied for the estimation of folic acid (FA) by cyclic voltammetry, chronoamperometry, linear sweep voltammetry, and differential pulse voltammetry techniques. The influence of pH (7), scan rate (50 mV/s), supporting electrolyte (0.1 M KCl) and FA concentration has been optimized. FA is precisely determined in the range from 0.01 to 1.5 µΜ with a low detection limit (0.002 µΜ), a low quantitation limit (0.068 µΜ), high reproducibility (RSD 0.37, 10 measurements), and high stability (98% activity after 50 days). FA in food samples was determined by the new sensor with high recoveries from 93 to 108.8%.
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