Herein, we report a novel sensor to detect trypsin using a purpose-designed fluorescein-labelled peptide with negatively charged carbon nanoparticles (CNPs) modified by acid oxidation. The fluorescence of the fluorescein-labelled peptide was quenched by CNPs. The sensor reacted with trypsin to cleave the peptide, resulting in the release of the dye moiety and a substantial increase in fluorescence intensity, which was dose-and time-dependent, and trypsin could be quantified accordingly. Correspondingly, the biosensor has led to the development of a convenient and efficient fluorescent method to measure trypsin activity, with a detection limit of 0.7μg/mL. The method allows rapid determination of trypsin activity in the normal and acute pancreatitis range, suitable for point-of-care testing. Furthermore, the applicability of the method has been demonstrated by detecting trypsin in spiked urine samples.