Ultrasensitive, reliable, and prompt prewarning of glyphosate (GLY) residue is essential to ensure environmental and food safety. Hence, a novel fluorescent sensor, N’-(7-(diethylamino)-2-oxo-2H-chromen-3-yl)methylene)-2-hydroxy-4-methoxybenzohydrazide (DCB), is proposed. It shows a significant and specific fluorescence quenching response to Cu2+ and forms a Cu2+ complex (DCS-Cu2+). More importantly, GLY can bind to Cu2+ more strongly than DCB, and the DCB-Cu2+ complex releases free DCB through competitive coordination. Thus, GLY can restore the fluorescence of DCB which has been extinguished by Cu2+. Through fluorescence enhancement, the DCB-Cu2+ complex shows a quick, specific, and sensitive response to GLY, and a good linear relationship between fluorescence intensities and GLY concentrations is found in the range of 0–11 μM with a limit of detection of 0.28 μM (47.34 ng/mL). Furthermore, the suggested sensor has been successfully used for the detection of GLY in actual samples.