Abstract: The binding characteristics between ponceau 4R and trypsin was investigated by fluorescence, UV-vis absorption, Fourier transform infrared ( FT-IR) and circular dichroism ( CD) spectroscopy, to obtain the binding constant, binding sites ( n) , the types of forces, the binding distance ( r) between ponceau 4R and trypsin, and other information. The results showed that ponceau 4R had a strong ability to quench the intrinsic fluorescence of trypsin, the quenching mechanism of trypsin by the ponceau 4R was a static process. The calculated thermodynamic parameters showed that the binding process was primarily driven by hydrogen bonds and van der Waals forces.The binding constant between them was 104 orders of magnitude, revealing that ponceau 4R can bind to trypsin with moderate affinity.Moreover, analysis of synchronous fluorescence, UV-vis absorption, Fourier transform infrared ( FT-IR) and CD spectra demonstrated that the binding interaction induced the microenvironment changes and conformational alteration of trypsin with increases in α-helix and random coil contents, and reduction of β-sheet and β-turn structures, resulting in partial shrinkage of the polypeptides of protein.