The supramolecular interactions among thioflavin T (ThT), two herbicides paraquat (PQ) and diquat (DQ), and macrocyclic cucurbit[8]uril (Q[8]), were studied using spectrofluorimetry, ultraviolet-visible absorbance spectrometry, H nuclear magnetic resonance spectroscopy, and isothermal titration calorimetry. A new method based on fluorescence quenching of the fluorescent host-guest complexes of Q[8]-ThT (the probe) upon cooperative binding with PQ or DQ to form a ternary complex, was proposed for the analytical determination of the two herbicides in aqueous solutions. Detection limits of 7.95 × 10 mol L and 8.07 × 10 mol L were obtained for PQ and DQ, respectively. Recoveries obtained by the proposed method in real-world examples such as river water and cabbage extracts were 104-108%. Interestingly, this method demonstrated high selectivity towards PQ and DQ in the presence of various metal ions and quaternary ammonium substances. Such a method provides a rapid, selective, sensitive and facile strategy for herbicides detection and quantification.