Mycoplasma wenyonii is a wall-less hemotrophic prokaryote with worldwide distribution. This paper describes the development of a LAMP method targeting 16S rRNA for specific detection of M. wenyonii in its vectors and cattle. The LAMP method is specific for M. wenyonii detection and more sensitive than PCR. A total of 330 blood samples from cattle were tested by LAMP and PCR detection, 71 (21.5 %) samples were positive by the LAMP, while only 62 (18.8 %) were positive by PCR. For detecting transmission vectors, 26 lice, 30 flies, and 26 mosquitoes were collected and 18 lice, 20 flies, and 21 mosquitoes were tested positive by LAMP and PCR. These results indicate that the LAMP assay is a simple and convenient diagnostic tool for M. wenyonii detection and can be used in epidemiological surveys.