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The nitrate ion (NO₃⁻) is a typical pollutant in environmental samples, posing a threat to the aquatic ecosystem and human health. Therefore, rapid and accurate detection of NO₃⁻ is crucial for both the aquatic sciences and government regulations. Here we report the fabrication of an amino-functionalized, vertically ordered mesoporous silica film (NH₂-VMSF) confining localized copper nanoparticles (CuNPs) for the electrochemical detection of NO₃⁻. NH₂-VMSF-carrying amino groups possess an ordered perpendicular nanochannel structure and ultrasmall nanopores, enabling the confined growth of CuNPs through the electrodeposition method. The resulting CuNPs/NH₂-VMSF-modified indium tin oxide (ITO) electrode (CuNPs/NH₂-VMSF/ITO) combines the electrocatalytic reduction ability of CuNPs and the electrostatic attraction capacity of NH₂-VMSF towards NO₃⁻. Thus, it is a rapid and sensitive electrochemical method for the determination of NO₃⁻ with a wide linear detection range of 5.0–1000 μM and a low detection limit of 2.3 μM. Direct electrochemical detection of NO₃⁻ in water samples (tap water, lake water, seawater, and rainwater) with acceptable recoveries ranging from 97.8% to 109% was performed, demonstrating that the proposed CuNPs/NH₂-VMSF/ITO sensor has excellent reproducibility, regeneration, and anti-interference abilities.