Determination of protease activity is very important for disease diagnosis, drug development, and assuring quality and safety of dairy products. Therefore, the development of low-cost methods for assessing protease activity is critically essential. Here, we demonstrate acoustic wave-based biosensor operated in the thickness-shear mode (TSM) enables low-cost detection of protease activity in a real-time mode. The TSM sensor was based on a protein substrate (PS) β-casein immobilized on a piezoelectric quartz crystal electrode. The β-casein layer was immobilized onto a gold surface by carboxylate terminated self-assembled monolayer (SAM) of 11-mercaptoundecanoic acid (MUA). Activation of the carboxylic acid terminal was performed by reaction of a mixture of water-soluble N-(3-Dimethylaminopropyl)-N0-ethylcarbodiimide (EDC) and N–Hydroxysuccinimide (NHS) on the electrode surfaces. We demonstrated that β-casein can form stable assembly on a piezoelectric quartz crystal electrode. After enzymatic reaction with trypsin, it cleaved the surface-bound β-casein substrate, which increased the frequency of crystal in a sigmoidal manner. Trypsin was detected in the range of concentrations 1–50 nM. The limit of detection was 0.2 nM. Initial reaction rates measured at different enzyme concentrations have been used to construct a calibration curve. In consideration of results obtained we believe that the TSM biosensor is a useful tool for protease analysis.