Pepsin serves as a potential biomarker for laryngopharyngeal reflux disease (LPRD). The reported methods for detecting pepsin still have limitations, such as long reaction times and low sensitivity. Therefore, it is crucial to develop a rapid, simple and sensitive detection method. In this study, a fluorescence immunochromatographic test strip was designed for point-of-care diagnostics of LPRD.To obtain optimal fluorescence immunochromatographic conditions, key experimental factors, including microsphere particle size, binding pH, and resuspension dilution ratio, were systematically evaluated. The optimal fluorescent microsphere size was determined to be 300 nm, the most suitable reaction pH was 8.0, and the ideal resuspension solution to storage solution ratio was 1:4. Under this optimal conditions, the sensor had a linear range of 2.5-100.0 ng/mL and a detection limit of 1.9 ng/mL. The relative standard deviation was below 15% for both intra and inter batch reproducibility. Under the condition of 37℃accelerated damage, the test value of the test strip could be kept basically unchanged for 14 days. There was no cross-reactivity of the test strip with pepsinogen I, pepsinogen II and other interferents. In the actual sample detection, the recovery rate was between 85% and 115%. Furthermore, the proposed method was demonstrated to be green and strong applicability based on the evaluation of the AGREEprep and the BAGI. In addition, the method has the advantages of high sensitivity, accuracy, green and applicability, which can meet the needs of rapid quantitative on-site detection.