Ellipsometry and total internal reflectance fluorescence spectroscopy (TIRF) have been employed to investigate the layer structure of gelatin adsorbed from aqueous solutions onto silica/glass and methylated silica/glass, as well as the effects of addition of the proteolytic enzymes krillase and trypsin, in relation to temperature, enzyme concentration and enzymatic activity. The results for the hydrophilic substrates show that homogeneous and heterogeneous exchange occurs readily, as does autolysis of trypsin at the interface. At the hydrophobic substrates, the effect of exchange is limited and a residual gelatin fraction is present at the interface throughout. The interfacial behavior of gelatin above and below the helix formation temperature (Thelix) shows that more extended surface layers are formed at both substrates below Thelix . At the hydrophilic substrates, the higher adsorbed layer thickness below Thelix is mainly due to the adsorption of more gelatin than at the higher temperature, whereas, at the hydrophobic substrates, the increase in layer thickness below Thelix is due to a decrease in packing density. Enzyme addition to preadsorbed gelatin at methylated silica results in the transition to a thinner and denser layer, that contains both residual gelatin and proteolytic enzymes (i.e., krillase or trypsin). At hydrophobic surfaces, a faster and more extensive degradation of the gelatin layer is observed with increasing krillase concentration, the effect of which is similar above and below Thelix . The effect of trypsin addition to preadsorbed gelatin is enhanced at TThelix . Finally, the exposure of preadsorbed gelatin to inactivated krillase showed a nearly complete elimination in the effects observed upon addition of intact krillase. This indicated that the enzymatic activity of krillase in its native form plays a major role for the interaction between krillase and preadsorbed gelatin.