The supramolecular interaction between β-cyclodextrin and brodifacoum, an anticoagulant rodenticide of the second generation, was studied by spectroscopy. The results showed that brodifacoum and β-cyclodextrin could form an inclusion complex with an association constant of 1.048×104 L·mol-1 and a 1∶1 stoichiometry based on Benesi-Hildebrand equation. The inclusion mechanism was proposed to explain the inclusion mode. It was indicated that the hydrophobic group of brodifacoum molecule, biphenyl, entered into the cavity of β-cyclodextrin. At the same time, it was also observed the significant enhancement of fluorescence of brodifacoum after forming inclusion complex. According to the fluorescence enhancement phenomenon, a spectrofluorimetric method of detecting brodifacoum in aqueous media was established with the linear range of 8.0×10-8-4.0×10-6 mol·L-1 and the correlation coefficient of 0.999 4. The detection limit of the method was 8.8×10-9 mol·L-1. The proposed method was successfully applied to determine the trace amount of brodifacoum in environment water and the recovery was in the range of 87.3% to 103.9%.