Objective To establish a bacterial outer membrane vesicle labeling method based on luciferase reporting system. Methods Using the characteristics of high abundance outer membrane protein on the surface of bacterial outer membrane vesicles, the outer membrane protein-luciferase fusion protein was constructed to realize the " Self-luminescent" of bacterial outer membrane vesicles for detection. Results The luciferase enzyme markers of bacterial outer membrane vesicles were constructed by constructing OmpA-NanoLuc fusion protein, and the outer membrane vesicles secreted by bacteria expressing the fusion protein had definite luciferase activity, the bacterial outer membrane vesicles could be detected by detecting the activity of fluorescein enzyme. Conclusion A bacterial outer membrane vesicle labeling method based on fluorescein enzyme reporting gene was successfully constructed, and the bacterial outer membrane vesicles could be detected without the use of cell membrane dyes.