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An electrochemical sensor based on silver nanoparticles (AgNPs) was developed by immobilizing dissolved organic matter (DOM) to interact with AgNPs on a glassy carbon electrode (GC), forming a DOM/AgNP/GC composite. AgNPs have been fabricated and characterized using ultraviolet−visible spectroscopy, fluorescence spectroscopy, and transmission electron microscopy. The immobilization efficiency and stability of the DOM/AgNP/GC composite have been optimized through studying the effects of electrode material type, immobilization technique, sticking duration, supporting electrolyte, and pH using square wave anodic stripping voltammetry. The detection of atrazine (Atz), a common herbicide, in aqueous systems has a great significance because of its toxicity to humans and other animals. The DOM/AgNP/GC composite has been used for Atz assessment under the optimized conditions based on the aggregated and nonaggregated AgNPs. The sensor linear range is between 20 and 220 μg/L for both aggregated and nonaggregated AgNP-based sensors. Atz could also be detected on the basis of its electrochemical oxidation at DOM/AgNP/GC with a linear range of 10−140 μg/L. Both sensors have been employed for the assessment of Atz in natural water with acceptable recovery values.