Monitoring nitrite in water and food is critical for ensuring human health. Herein, an electrochemical sensor was constructed for determining nitrite based on iodide probe and gold nanoparticles-poly(L-arginine)-poly(L-lysine) (AuNPs-PArg-PLys) modified electrode. The AuNPs-PArg-PLys nanocomposites were fabricated by one-step in-situ electrochemical synthesis method. Electrochemical detection of nitrite was carried out using square-wave voltammetry. With the assistance of iodide probe, the minimum detectable nitrite concentration reduced remarkably by five orders of magnitude when compared to that without this probe. The iodide-assisted AuNPs-PArg-PLys-based electrochemical sensor could determine nitrite from 1.0 nM to 10.0 μM with a detection limit of 271.8 pM. This sensor possessed the advantages of simplicity, rapidness, low detection limit, wide response range, excellent reproducibility, and high selectivity towards nitrite, which was successfully utilized to measure nanomolar-scale nitrite in tap water and beef. The proposed sensor offered potential applications in environmental protection and food safety.