Trypsin is a specific and reliable diagnostic biomarker of pancreatic function and pathological changes. Thus, detecting trypsin and screening trypsin inhibitors are highly important for clinical diagnosis and disease treatment. Herein, we propose a colorimetric/fluorescence sensing probe based on carbon quantum dots (O-CDs) and cytochrome C (Cyt C) for sensitive and specific trypsin detection and trypsin inhibitor screening. The prepared O-CDs exhibit a persistent fluorescence and environmental tolerance. In the presence of trypsin, Cyt C is hydrolyzed into a heme with oxidase mimetic activity that can convert colorless TMB into blue oxTMB, which strongly absorbs at 652 nm and quenches the fluorescence of the O-CDs at 580 nm via the inner filter effect. This sensing probe realizes the precise determination of trypsin with a wide linear range (10-2000 ng/mL in both colorimetric and fluorescence modes) and a low limit of detection (0.05 ng/mL in colorimetric mode and 3.44 ng/mL in fluorescence mode). The developed method was then applied to detect trypsin in human serum and urine with good accuracy and reproducibility. Furthermore, the sensing probe was successfully applied to screen trypsin inhibitors from legumes on the basis of their ability to inhibit the trypsin activity. Thus, this dual-mode detection method has great potential for clinical application in the diagnosis of trypsin-related diseases, as well as for screening trypsin inhibitor drugs.