To establish a ratiometric fluorescence sensing system for the detection of glyphosate, nitrogen-sulfur doped carbon quantum dots nano-fluorescent probe was prepared by one-step hydrothermal method. At an excitation wavelength of 360 nm, the emission peak with maximum fluorescence intensity was obtained at 470 nm. The generation of 2.3-diaminophenazine by o-phenylenediamine oxidation produces a new fluorescence emission peak at 570 nm and a decrease in fluorescence at 470 nm. The fluorescence peaks at 470 and 570 nm form a ratio state. In the presence of glyphosate, reduces the oxidation of 2.3-diaminophenazine, this results in enhanced fluorescence at 470 nm and weakened fluorescence at 570 nm. Under the best conditions, the ratiometric fluorescence sensing system has good selectivity, the linear at glyphosate concentrations of 0.005–10 µg/mL, the limit of detection was 2.88 µg/kg, the limit of quantitation was 4.07 µg/kg. In addition, it is worth mentioning that this developed sensing system has shown good detection performance in 10 miscellaneous samples, the simple, efficient, and highly sensitive ratiometric fluorescence detection sensing strategy is provided.