Pepsinogen I in human serum is a common biomarker for diagnosing gastroesophageal reflux in clinical medicine. Traditional detection methods for pepsinogen I have the disadvantages of being time-consuming and having high cost and low sensitivity. In this study， a fluorescence detection system for the rapid and quantitative measurement of pepsinogen I is designed. First， a portable microscope is used to take a fluorescence image of the samples， and then the R channel image-processing algorithm of the RGB color mode is used to detect and analyze the images. Finally， according to experimental data， a standard curve is fitted with a fitting degree of 99.556%， and the variation coefficient of the test results is <4.9%. The experimental results show that the designed detection system can accurately and rapidly quantify pepsinogen I concentration.