Low level of trypsin has adverse impacts on digestion, and the obvious increase of trypsin may indicate the occurrence of pancreatitis or chronic renal failure. In addition, the secretory of trypsin outside of pancreatic tissue may involve a precursor to cancer. Trypsin concentration is closely related to life activities. Simple and timely monitoring of trypsin content can provide important reference value for disease diagnosis. Therefore, a sensitive and rapid fluorescent method was developed for determination of trypsin in urine based on carbon nitride quantum dots (CNQDs) and gold nanoclusters (AuNCs). CNQDs was synthesized via solvothermal treatment of bulk carbon nitride (C3N4) powder which was obtained by calcining melamine. The CNQDs displayed blue emission under radiation of UV light at 365 nm and the fluorescent band was at 440 nm. Albumin bovine serum (BSA) and CNQDs were used as reducing agents and stabilizers to prepare AuNCs which absorbed on the surface of CNQDs forming CNQD-AuNCs. CNQD-AuNCs with dual emission wavelengths at 440 and 650 nm displayed red fluorescence under radiation of UV light at 365 nm. BSA and AuNCs structure can be destroyed leading to aggregation of AuNCs in the presence of trypsin owing to the hydrolysis of BSA catalyzed by trypsin. Emission at 650 attributed to AuNCs is quenched and emission at 440 nm produced by CNQDs remain unchanged. The detection of trypsin can be performed by using fluorescent responses of CNQD-AuNCs. Fluorescent intensity at 650 nm gradually decreased with increasing trypsin concentration, while fluorescent intensity at 450 nm stayed unchanged. The ratio of fluorescent intensities at 650 and 440 nm had a perfect linear correlation with the concentrations of trypsin in the range of 10～400 ng·mL-1 with a good coefficient (R2=0997 6). The linear regression equation was y=2471～0004x, where x was the concentration of trypsin (ng·mL-1), and y represented ratio of intensity at 650 and 440 nm. Limit of detection (LOD) for trypsin was calculated to be 15 ng·mL-1 at a signal-to-noise ratio of 3. The concentration of trypsin in urine (the actual concentration was 50, 100 and 150 ng·mL-1) detected by this ratiometric method was 5241, 10325 and 15439 ng·mL-1, respectively. The recoveries of trypsin were 10293%～10482% with relative standard deviations of 357%～416%. AuNC@CNQDs nanosensor provide build-in self-calibration for correction of a variety of unfavorable factors by using the ratiometric responses as signals to detect trypsin. The ratiometric method can overcome shortcomings of signal response which is susceptible to effects of external factors such as light bleaching, nanosensor concentration, excitation light intensity and optical path, and so on. In summary, the developed method has been applied for detection of trypsin in urine with high sensitivity and selectivity, providing scientific basis for detection of trypsin in real application.