The supramolecular interaction of gemfibrozil and β-cyclodextrin has been studied by flow injection spectrofluorimetry. The results show that β-CD reacts with gemfibrozil to form a 1:1 complex with an apparent association constant of 7.57×102 L/mol. Based on the enhancement of the fluorescence intensity of gemfibrozil, a flow injection spectrofluorimetric method for the determination of gemfibrozil in bulk aqueous solution in the presence of β-CD was developed. The linear range was 0.032-70.00g/mL. The detection limit was 9.6ng/mL with a sampling rate of 80h−1. There was no interference from the excipients normally used in tablet formulations and the serum compositions. The proposed method was successfully applied to the determination of gemfibrozil in capsules and serum.