The work aims to establish a fluorescence-tracer liquid method to test the closure integrity of ribavirin eye drops packaging system, and provide a universal and cheap scheme for the closure integrity verification of eye drops packaging. With Rhodamine B as the tracer, high performance liquid chromatography-fluorescence detector was used to test and evaluate the sealability of ribavirin eye drops during shelf life and after simulated clinical opening. The method could accurately detect 5 μm aperture leakage in packaging materials, and the detection limit level was up to level 3 in "Technical Guide for Sealing Research of Packaging System for Chemical Injection" (For Trial Implementation), and effectively evaluate the closure integrity of packaging system for ribavirin eye drops. The method has strong versatility and high sensitivity, which can provide technical support for testing the closure integrity of eye drops packaging.