A collagen cross-linked with a pyrene derivative was prepared, and the gel structure of the heat-denatured collagen (gelatin) containing the chemical cross-links was examined using the fluorescence depolarization method. The fluorescence anisotropy of the cross-linked collagen decreased with an increase in temperature, suggesting a helix-to-coil transition. The denaturation temperature of the collagen was determined by the fluorescence anisotropy. The Perrin−Weber equation was applied to the analysis of the denaturation and regeneration of the collagen triple helix. The quantity of the triple helix regenerated from random coils was estimated from the Perrin−Weber plot. The helix content values in the regenerated cross-linked collagen obtained by the fluorescence anisotropy were different from those obtained by the circular dichroism. The models of the gelatin gel containing chemical cross-links were considered from the helix content data obtained from the different methods. Cross-links promoted the regeneration of the collagen triple helix. However, the triple helix was re-formed by avoiding the cross-link at the higher degree of cross-linking.