The interaction between three flavonoids, i.e., Luteolin (LTL), Quercetin (QCT), and Naringenin (NGN) and bovine lactoferrin (BLF) at pH 7.4 was investigated by fluorescence quenching spectra, synchronous fluorescence spectra, and UV-visible absorption spectra. The results indicate the fluorescence of BLF quenched by Luteolin (LTL), Quercetin (QCT), and Naringenin (NGN) via static quenching. The main force between QCT and LTL with BLF was van der Waals interactions and hydrogen bonds. Electrostatic interactions played a major role in the binding process of interaction between NGN and BLF. Synchronous fluorescence was used to study the conformational changes of BLF. The values of binding constant (Ka) and number of binding sites (n) at different temperatures (300K, 305K, 310K) were also calculated, respectively. The results of corresponding thermodynamic parameters as well as binding distance between BLF and LTL, QCT, or GNG were obtained. These results implied that Luteolin (LTL), Quercetin (QCT), and Naringenin (NGN) could provide important guides for compound quantity (e.g., medicine dosage) and the design of new compounds (or drugs).