Under simulated physiological conditions, the interaction between Tylosin Tartrate (TT) and Lysozyme (LYSO) was investigated at pH=7.40 by fluorescence spectroscopy. The results indicated that TT could strongly quench the intrinsic fluorescence of LYSO. By determining the quenching constants of the reaction between TT and LYSO at different temperatures, the quenching mechanism was proven to be a static quenching process. The thermodynamic parameters (ΔH, ΔS) of the reaction between TT and LYSO were obtained by the Van 't Hoff equation, and were 27.80 KJ mol-1 and 166.28 J mol-1 K-1, respectively. The results showed that hydrophobic interaction between TT and LSYO was dominant. Synchronous fluorescence spectra revealed that the conformation of LYSO was changed. This method could be applied to measure the content of TT.