The present research investigated the effect of dynamic high pressure microfluidization (DHPM) in low pressure ranges (60-100 MPa) on the molecular structure of papain with the help of fluorescence spectra as the detection method. The result showed that after the treatment of DHPM at 60-100 MPa, the fluorescence intensity of papain, tryptophan (Trp) and tyrosine (Tyr) residues all decreased to different extents. Meanwhile, with the increase in the treatment pressure, their fluorescence intensity would gradually increase and the fluorescence emission peak would gradually red shift from 334 and 277.5 nm before treatment to 335 and 278 nm after 100 MPa treatment for papain and Trp residue respectively; after the treatment, with the samples being placed at 0-4 ℃ for 24 h, the fluorescence spectra of papain, Tyr and Trp residues in various experiment groups basically maintained the same changing tendency compared to that of newly treated samples. Hence, it showed that after the treatment of DHPM in low pressure ranges, the Trp residue of papain was gradually brought to light and formed new and comparatively stable molecular conformation.