Herein, a sensing platform for the rapid quantitative detection of glyphosate with high sensitivity and selectivity was constructed by enzyme-free ratiometric fluorescence and colorimetric dual read-out assay based on ultrathin g-C3N4 nanosheets (UCN NS).Fluorescent diaminophenylazine (DAP) with fluorescence was from the oxidation of o-phenylenediamine (OPD) in the presence of Cu2+. DAP could inspire a significant fluorescence quenching of nanosheets due to photo-induced electron transfer (PET), therefore exhibiting a ratiometric fluorescence response. In the presence of glyphosate, the strong chelation of Cu2+ by glyphosate inhibited the oxidation of OPD, which reduced the fluorescence intensity of DAP, while the fluorescence of the ultrathin nanosheets was restored. Moreover, the oxidation of OPD (colorless) to DAP (yellow) was a distinctly visible color change process, which provides well-suited conditions for colorimetric analysis. At the optimum experimental conditions, the detection concentration range of glyphosate was 0.001–0.48 μg/mL, and the limit of fluorometric detection was 0.49 ng/mL (S/N = 3). This assay was simple and easy to use for the detection of glyphosate in food safety and the environment with satisfactory results.