Recombinant proteins expressed by prokaryotic expression system are normally in the form of inclusion. In the present paper, refolding process of recombinant pan-allergen profilin protein induced by urea has been investigated by using circular dichroism spectra, fluorescence spectra, synchronous fluorescence spectra systematically. And the spectral characteristics of the renaturation were obtained. In addition, bioinformatics methods including predications of secondary and tertiary structures have also been used to explain the spectral characteristics and analyze the conformational changes of the protein during renaturation in vitro. Results from this study should be useful to the establishment of a spectral method examining the extent of protein renaturation, and be helpful to the understanding of the mechanism of renaturation of recombinant protein.