Theileria annulata (T. annulata), the causative agent of tropical theileriosis, is a protozoan parasite that also causes lymphoproliferative diseases in cattle. Development of reliable and fast methods are necessary in the epidemiological investigation of T. annulata in ticks and animals. Real-time PCR possesses merits of rapidity, accuracy, reliability, automation and ease of standardization, which is widely used for the detection of blood borne parasites. In this study, species-specific primers and TaqMan probe were designed on the basis of the 18s rRNA gene sequence of T. annulata, and the real-time PCR assay was developed by optimizing the reaction parameter. The performance of real-time PCR was assessed by testing 47 blood samples from cattle and comparing with the results from conventional PCR. The results show that this real-time PCR assay could specifically detect 10 copies DNA of T. annulata, which is 10-fold sensitivity more than conventional PCR. No cross-reactions were observed with Babesia bovis, Babesia bigemina, Trypanosoma evansi and Theileria equi. Of the 47 field samples collected from Xinjiang Uygur Autonomous Region, China, 36.17% were detected by real-time PCR, and 25.53% were found positive for T. annulata infection by conventional PCR. These results indicated that the real-time PCR assay is a useful approach for detecting T. annulata infections and has potential as an alternative tool for ecological and epidemiological surveillance of ovine theileriosis.