Bartonella melophagi and Trypanosoma melophagium are intracellular bacteria and blood protozoan parasites, respectively, that can be transmitted by Melophagus ovinus and pose risks to human and animal health. Eight pupae and 214 M. ovinus were collected from three sampling sites in southern Xinjiang, China, between March 2019 and June 2023. The morphological characteristics and 18S rRNA sequences were used to identify M. ovinus. The gltA gene was used to detect B. melophagi and the 18S rRNA gene was used to detect T. melophagium in all samples. The DNA of B. melophagi was detected in 214 (100.00%, 214/214) samples and the DNA of T. melophagium was detected in 101 (47.20%, 101/214) samples using PCR and sequencing. Eight pupae produced two different outcomes: B. melophagi was positive and T. melophagium was negative. This study presents conclusive evidence for the presence of B. melophagi and T. melophagium in M. ovinus. To the best of our knowledge, this is the first report of T. melophagium in M. ovinus in China. These findings offer a significant reference for the advancement of the knowledge and prevention of M. ovinus, B. melophagi, and T. melophagium in China.