An amperometric biosensor for the detection of trypsin was developed. The latter was based on a two-layer configuration, namely, a polymer-glucose oxidase inner layer and a gelatin outer layer. In the presence of glucose, the enzyme layer produces H2O2 and hence an amperometric signal due to H2O2 electrooxidation was generated by potentiostating the electrode at 0.6 V. The biosensor detects the change in the increase in the maximum current caused by the proteolytic digestion of gelatin, which covers the platinum electrodes, thereby facilitating a speedier access for the glucose substrate to the electrode modified with both poly(pyrrole-alkylammonium) and glucose oxidase molecules. Our biosensor detected low trypsin concentrations down to 42 pM with a response time of approximately 10 min, making it a very sensitive device in the detection of lower trypsin levels with such future putative applications as the diagnosis of pancreatic diseases.