Abstract: Objective To develop a sensitive and specific real time reverse transcription PCR (RT-PCR) assay for nucleic acid detection of novel Bunyavirus. Methods Primers and TaqMan probe were designed according to the L gene of novel Bunyavirus and the real time RT-PCR was used to detect novel Bunyavirus from serum and plasma samples of 50 clinical suspected patients and from serum of 25 healthy people. Two virus strains isolated from the patients’ serum were used to evaluate the specificity and sensitivity as well as the reproducibility of the established real time RT-PCR assay. Results The established real time RT-PCR assay had high specificity, with the Ct values showing good linear relation to the copy numbers of template DNA in the standard curve (R2=0.999). In this assay, the target gene could be detected if the template DNA is more than 25 copies in 25 μl PCR reaction, indicating that the sensitive detection limit was 1 copy/μl. The reproducibility tests indicated that the intraassay coefficient of variation were all lower than 1.0%. Among the 50 patients’ serum samples, 30 were positive, 25 control’s serum samples and 28 rats lung and testes samples were all negative. Two of 8 blood samples were positive in virus culture. Conclusion The established real time RT-PCR assay was with high specificity, sensitivity and reproducibility, which could be used in the rapid nucleic acid detection of novel Bunyavirus in humans and animals and applied in the preliminary identification of the virus after isolation and culture.