Rapid and sensitive determination of nitrate (NO3−) is crucial as NO3− contamination in aquatic environments poses increasing risks to ecosystem stability and drinking water safety. Herein, an electrochemical NO3− sensor was reported based on cuprous oxide (Cu2O) nanoparticles integrated with carboxylated multiwalled carbon nanotubes (cMWCNTs) on indium tin oxide (ITO) electrode. The sensing interface was constructed by drop-casting conductive cMWCNTs network followed by in situ electrodeposition of Cu2O nanoparticles. The cMWCNT scaffold improved interfacial charge transport, enlarged the accessible surface area, and promoted the homogeneous distribution of Cu2O, while Cu2O acted as nanocatalyst for NO3− electroreduction. Electrochemical characterization confirmed that Cu2O@cMWCNTs/ITO electrode exhibited lower charge-transfer resistance and stronger NO3− reduction current than Cu2O modified ITO electrode. Under optimized conditions, the fabricated sensor enabled NO3− detection over two linear ranges of 1–100 and 100–800 μM, with limit of detection of 0.79 μM. The sensor showed good tolerance toward dissolved oxygen and common inorganic ions, as well as satisfactory fabrication reproducibility. NO3− detection in seawater was also achieved. This simple and effective Cu2O-carbon nanotube interface showed potential for electrochemical monitoring of NO3− in environmental waters.