A luminescent G-quadruplex-derived detection technique was established to monitor glyphosate (GLY) using a phosphorescent iridium(III)-based probe. By exploiting the long lifetime of the probe, the use of time-resolved emission spectroscopy (TRES) could overcome substantial fluorescence background interference. This platform displayed excellent sensitivity for GLY with a limit of detection of 26.4 nM in Tris buffer, and 79.3 nM in Tris buffer containing soybean extract to simulate a complex environment. Due to the specificity of the GLY aptamer, the platform could selectively recognize GLY in complex samples. The feasibility of the method was evaluated by analysis of licorice, ginseng, ginger, and soybean samples, as well as in HUVEC and LO2 cell extracts, with good recovery values ranging from 85 % to 110 %. Importantly, GLY excreted by mouse fed with GLY could also be rapidly and efficiently detected with high correlation with a commercial kit, indicating the reliability of this platform for the rapid and efficient monitoring GLY in animals exposed to GLY. Finally, besides being a rapid and efficient detection method, this method also has the potential for rapid diagnosis at the point of care.