Proteolysis of milk proteins, such as caseins, caused by milk proteases, can change the organoleptic and nutritional characteristics of milk, and therefore it is essential to monitor this enzymatic activity. We used trypsin as a model protease because of its role as a biomarker for pancreatitis. The aim of this work was to demonstrate the detection of proteolysis of β-casein by trypsin using a multiharmonic quartz crystal microbalance (QCM) biosensor. The β-casein layer was deposited from a 0.1 mg mL-1 solution on a hydrophobic surface consisting of a self-assembled monolayer of 1-dodecanethiol on the gold electrode of the QCM. The addition of an increasing concentration of trypsin leads to the removal of the casein layer due to proteolysis, and correlates with an increase in the resonant frequency of the QCM. We investigated the effect of trypsin concentrations (0.3-20 nM) on the kinetics of the proteolysis of β-casein and demonstrated that the frequency increase is proportional to the protease concentration. Consequently, an inverse Michaelis-Menten model was used to estimate the Michaelis-Menten constant (KM = 0.38 ± 0.02 nM) and the limit of detection (LOD = 0.16 ± 0.02 nM). The thickness, mass and viscoelastic properties of the protein adlayer after its formation and following the proteolytic cleavage were evaluated by means of multi-harmonic analysis. We found that β-casein is preferably adsorbed on the hydrophobic surfaces as an asymmetrical double layer, of which the innermost layer was found to be denser and thinner (about 2.37 nm) and the outermost layer was found to be less tightly bound and thicker (about 3.5 nm).