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Glyphosate is a broad-spectrum pesticide used in crops and is found in many products used by industry and consumers. Unfortunately, glyphosate has been shown to have some toxicity toward many organisms found in our ecosystems and has been reported to have carcinogenic effects on humans. Hence, there is a need to develop novel nanosensors that are more sensitive and facile and permit rapid detection. Current optical-based assays are limited as they rely on changes in signal intensity, which can be affected by multiple factors in the sample. Herein, we report the development of a dual emissive carbon dot (CD) system that can be used to optically detect glyphosate pesticides in water at different pH levels. The fluorescent CDs emit blue and red fluorescence, which we exploit as a ratiometric self-referencing assay. We observe red fluorescence quenching with increasing concentrations of glyphosate in the solution, ascribed to the interaction of the glyphosate pesticide with the CD surface. The blue fluorescence remains unaffected and serves as a reference in this ratiometric approach. Using fluorescence quenching assays, a ratiometric response is observed in the ppm range with detection limits as low as 0.03 ppm. Our CDs can be used to detect other pesticides and contaminants in water, as cost-effective and simple environmental nanosensors.