Carbon dots (CDs) having low cost and low toxicity and synthesized via a green route were applied to establish a fluorescent nanoprobe for the measurement of glyphosate. The synthesis was realized via a one-pot hydrothermal procedure using Sophora japonica leaves as the carbon source. It was found that electron transfer occurred between Fe3+ and the as-prepared CDs. Therefore, Fe3+ exhibited a specific dynamic-quenching toward CDs. However, the electron transfer process was inhibited by glyphosate. The fluorescence of the quenched CDs/Fe3+ system was recovered by the addition of glyphosate. It resulted from the strong complexation between Fe3+ and the functional groups (like -PO3H2 and -COOH) in the glyphosate molecule. These functional groups captured Fe3+ from the CD/Fe3+ system to reduce the electron transfer. With such a design, the rapid detection of glyphosate could be realized by this turn-on fluorescent sensor based on the CD/Fe3+ system. Under optimal conditions, the CD/Fe3+ system showed a concentration-dependent fluorescent response toward glyphosate in the linear range from 0.1 to 16 ppm. The limit of detection was calculated to be as low as 8.75 ppb (3σ/S). In addition, the successful detection of glyphosate in real samples with satisfactory recoveries exhibited a practical application of the CD/Fe3+ nanoprobe in food safety and environmental monitoring.