Casein films seem to be a promising substrate for the study of the enzymatic activity of trypsin and the detection of its concentration. In the first part of this work, a multilayer was formed with the layer by layer process and its characterization was carried out by multiharmonic Quartz Crystal Microbalance (QCM). The casein aggregation mechanism was investigated by allowing the casein amphipathic monomers to absorb at low concentration solutions (0.1 mg/mL) through hydrophobic interactions on a hydrophobic surface consisting of a self-assembled 1-dodecanthiol monolayer on a gold electrode of the quartz crystal. In this procedure, the negative phosphoseryl clusters of the protein are free, and a subsequent incubation with calcium ions can make the positive layer capable of binding more casein molecules, forming an oligolayer. The possibility of carrying out the construction step-by-step of multilayer casein architectures was investigated, reiterating the casein-calcium-casein ion incubation process, since cations increase the surface hydrophobicity of the protein. In the second part, the possibility of using this architecture for an analysis of trypsin protease detection is investigated. After the enzymatic reaction, an increase in the resonance frequency of the piezoelectric crystal occurs, caused by a mass removal from the surface of the electrode, and it may be related to the concentration of the enzyme. All analyzes were carried out in a multimodal version, analyzing the construction of the multilayer and the detection of the enzyme, with multiple resonant frequencies (from the first to the seventeenth). Finally, the possibility of building a multilayer of casein doped with gold nanoparticles was also studied, in order to obtain an increase in sensitivity in the detection of trypsin.