Glyphosate has excellent herbicidal activity, and its extensive use may induce residue in the environment and enter into humans living through the food chain, causing negative impact. Here, water-soluble 1.55 nm size nitrogen-doped carbon quantum dots (NCDs) with strong blue fluorescence were synthesized using sodium citrate and adenine. The maximum excitation and emission wavelengths of NCDs were 380 nm and 440 nm, respectively. The above synthesized NCDs were first used for the construction of a fluorescence sensor for glyphosate detection. It was found that Cu2+could quench the fluorescence of NCDs effectively through the photoinduced electron transfer (PET) process, which was confirmed using fluorescence lifetime measurements. Additionally, the fluorescence was restored with the addition of glyphosate. Hence, a sensitive turn-on fluorescence sensor based on NCDs/Cu2+for glyphosate analysis was developed. The LODs of glyphosate for water and rice samples were recorded as 0.021 μg/mL and 0.049 μg/mL, respectively. The sensor was applied successfully for ultrasensitive and selective detection of glyphosate in environmental water and rice samples with satisfied recoveries from 82.1% to 113.0% using a simple sample pretreatment technique. The proposed strategy can provide a significant potential for monitoring glyphosate residue in water and agricultural product samples.