Objective To investigate the pathogens carried by flies at Tibetan ports, and to provide a technical support for the health quarantine and law enforcement at ports. Methods From May to October, 2017, pathogen detection was performed for flies collected at Lhasa Gonggar International Airport, Jilong port, and Yadong port. Bacterial detection was performed for fly samples using the microbiological isolation and pure culture technology, 16S rRNA gene sequence identification, and physiological and biochemical identification techniques, and RT-PCR was used to detect the virus carried by flies. Results A total of 37 pathogenic bacteria (conditioned pathogens) were isolated from flies, including Enterococcus faecalis, Hafnia alvei, Moellerella wisconsensis, Escherichia coli, Proteus vulgaris, and Enterococcus durans. Virus detection found one case of rotavirus (group A), resulting in a detection rate of 2.0% (1/50). Conclusion Flies carry a large number of bacteria, and some of them may also carry enterovirus, which may lead to the risk of disease spread at frontier ports. Health quarantine departments should strengthen the monitoring and detection of flies at frontier ports to prevent the spread of infectious diseases.