A novel and sensitive fluorescent probe based on Cu2+-modulated polydihydroxyphenylalanine nanoparticles (PDOAs) has been developed for the detection of glyphosate pesticides. Compared to conventional instrumental analysis techniques, fluorometric methods have obtained good results in the field of agricultural residue detection. However, most of the fluorescent chemosensors reported still have some limitations, such as long response times, the high limit of detection, and complex synthetic procedures. In this paper, a novel and sensitive fluorescent probe based on Cu2+ modulated polydihydroxyphenylalanine nanoparticles (PDOAs) has been developed for the detection of glyphosate pesticides. The fluorescence of PDOAs can be effectively quenched by Cu2+ through the dynamic quenching process, which was confirmed by the time-resolved fluorescence lifetime analysis. In the presence of glyphosate, the fluorescence of the PDOAs-Cu2+ system can be effectively recovered due to the higher affinity of glyphosate for Cu2+, and thus released the individual PDOAs. Due to the admirable properties such as high selectivity to glyphosate pesticide, “turn on” fluorescence response, and ultralow detection limit of 1.8 nM, the proposed method has been successfully applied for the determination of glyphosate in environmental water samples.