A simple and sensitive electrochemical method was developed for the determination of trypsin by employing a specific heptapeptide (CRRRRRR) as a substrate. The positively charged heptapeptide substrate is self-assembled on the surface of a gold electrode through the thiol group of the cysteine (C) at the end of the peptide, which prevents the electrochemical probe [the ruthenium(III) hexammine complex] to access the electrode. The substrate peptide is hydrolyzed by trypsin, and this causes the fragments to leave the electrode and, consequently, the electrochemical signal to increase. The results show that the increase in the square wave voltammetric current of the ruthenium probe is linearly related to the activity of trypsin in the range from 0.0047 to 0.052 U·mL−1 with a detection limit of 0.0012 U·mL−1. This work demonstrates that the enzymatic cleavage of the substrate can be directly converted to an electrical signal to provide a simple and sensitive method for the determination of trypsin.