Trypsin is an enzyme that is included in the hydrolysis class of enzymes. Trypsin catalyzes the cleavage of proteins from a serine residue and is found in the digestive system of vertebrates. The cystic fibrosis, which is an autosomal recessive disorder, is associated with the trypsin deficiency. The meconium ileus is caused by this deficiency. Beside the clinical significance of trypsin, it is also used in many industrial applications, such as tissue culture labs, milk industry, proteomics, and different kinds of food production processes. In this study, a glucose oxidase based electrochemical biosensing system was constructed for sensitive and accurate determination of trypsin activity in different samples. A novel working principle was operated in the reported biosensor, which is based on the trypsin degradation ratio in the bioactive layer of the biosensor. Detailed optimization and characterization experiments were carried out to obtain the best electrochemical signals. The system introduced a linear determination range for trypsin activity between 6.65 and 33.25 U/ml. The biosensor was also used in the analysis of the trypsin activities of real samples.