This research put forward a simple and rapid fluorescence probe method based on competition strategy to detect homocysteine. The host-guest interaction behavior between cucurbit[7]uril and homocysteine was further studied via UV-vis spectroscopy, Fourier transform infrared spectroscopy, molecular modeling, and 1H NMR spectroscopy in aqueous solution. The binding constant of cucurbit[7]uril-homocysteine measured by 1H NMR titration is 2.40 ´ 103 L mol-1. Under the optimal detection conditions, the fluorescence intensity of the cucurbit[7]uril-berberine complex declined linearly with increasing concentration of homocysteine in the range from 5 to 200 µM, and the limit of detection was 1.76 µM. This established fluorescent probe method exhibits high selectivity for coexisting interfering substances. Meanwhile, it was also successfully employed to detect homocysteine in serum samples with the recovery rate of 85.1% to 102.0%. This supramolecular fluorescent probe based on competitive strategy provides a simple and fast method for the determination of homocysteine in practical samples.