A combination of spectral experiment and molecular modeling techniques was used to characterize the binding mechanism between enrofloxacin（EFLX）and bovine lactoferrin（BLF）.The binding constants，energy transfer paremeters and thermodynamic parameters were investigated.The influence of EFLX on molecular conformation of BLF and the molecular models of EFLX-BLF were observed and established.The results showed that dynamic quenching exited between EFLX and BLF with moderate bond.The value of binding distances（r） was low，indicating the occurrence of energy transfer.The drug had conformation effect on BLF，which resulted in the decrease of hydrophobic environment of the binding domain in BLF.According to the obtained thermodynamic parameters，the main interaction forces between EFLX and BLF were hydrogen bonds and Van der Waals forces.The results of molecular modeling revealed that hydrogen bonds and Van der Waals forces are the main binding forces in the EFLX-BLF system，combining with hydrophobic interactions.Molecular modeling was agreed in the experiment results，which provided helpful reference for the study of molecular reaction mechanism of the quinolones binding with bovine lactoferrin.