In this study, we developed a simple fluorescence sensing system to rapidly measure the total carbonyl stress in urine because carbonyls may form advanced glycation end products, leading to diseases. The traditional method for detecting carbonyl stress has been high-performance liquid chromatography (HPLC), which detects glyoxal, methylglyoxal, and 3-deoxyglucosone contents. The excitation and emission wavelengths used in the sensing system to monitor aldehyde content in urine were 340 and 500 nm, respectively. Rat urine samples were derivatized with 5,6-diamino-2,4-hydroxypyrimidine sulfate dihydrate. A 20 µL sample was added to a carrier composed of polydimethylsiloxane for testing, and only 1 s was required for detection. According to the fluorescence intensity results, the calibration curve for the total content of all aldehydes in the urine had a good degree of linearity with the fluorescence intensity (R2 = 0.9879). In addition, the urine of diabetic and healthy rats was tested, and the correlation coefficient between the total carbonyl content detected by the simple fluorescence system and HPLC reached 0.8132. Collectively, our findings demonstrated that our system can rapidly measure total carbonyl stress in urine with a small sample volume.