Under simulated physiological conditions, we studied the reactionmechanismofMethylGreen with Bovine serumalbumin at different temperature (293K, 303K, 310K) by utilizing fluorescence quenchingmethod and a new method (synchronous fluorescence), respectively. The results indicate that Methyl Green could quench the intrinsic fluorescence of Bovine serum albumin strongly, and the quenching mechanism was a static quenching process. The hydrogen bonding and van derWaals force played an important role on the conjugation reaction between Methyl Green and Bovine serumalbumin. The order ofmagnitude of binding constants (Ka) was 104, the number of binding site (n) in the binary system was approximately equal to 1 and the primary binding for Hydrogen bonding and van der Waals force were located at the structure domain II A of Bovine serum albumin. The results obtained by the two methods were consistent, which indicated synchronous fluorescence spectroscopy can replace traditional fluorescence quenching method to study reaction mechanismof dyes with proteins.