Electrochemical analysis of ascorbic acid (AsA) in physiological condition using a new hybrid film modified electrode is described. Electrochemical polymerization of luminol in 0.1 M H₂SO₄ solution was carried out using ZnO nanoparticles (ZnO-NPs) coated glassy carbon electrode (GCE) as working electrode. This hybrid film coated electrode noted as poly(luminol)/ZnO-NPs hybrid film modified GCE (PLu/ZnO-NPs/GCE). The atomic force microscope (AFM) and scanning electron microscope (SEM) studies were demonstrated that PLu/ZnO-NPs hybrid film covered the electrode surface and the ZnO-NPs particle sizes were <100 nm. The visible blue colored organic–inorganic (PLu/ZnO-NPs) hybrid films were observed on the electrode surface. Electrochemical studies proved that PLu/ZnO-NPs hybrid film modified electrode is electroactive in the pH range from 1 to 11 and the poly(luminol) (PLu) redox peak was pH dependent with a slope of −53 mV/pH. The PLu/ZnO-NPs modified electrodes electroactivity also investigated by catalyzing the oxidation of AsA, demonstrating its great potential applications in electroanalysis of AsA. The resulting, AsA electrochemical sensor exhibited a wide linear response range (from 1 × 10⁻⁶ to 3.6 × 10⁻⁴ M, r² = 0.9989), lower detection limit (1 × 10⁻⁶ M) and fast response time (3 s) for AsA determination. Our results show that PLu/ZnO-NPs hybrid film provides a novel and efficient platform for the oxidation of AsA and realizing efficient electrocatalysis and that the materials have potential applications in the fabrication of electrochemical sensors. Analysis of commercial vitamin C samples using PLu/ZnO-NPs hybrid film modified electrode was demonstrated and the obtained results are good agreement with the labeled amount.