Abstract: To establish a reverse transcription droplet digital PCR (RT-ddPCR) detection method of Nipah virus (NIV), a recombinant plasmid was constructed using the conserved sequence of the NIV-N gene as the target gene. The plasmid was used as template to generate in virto transcription RNA, and five sets of specific primer probes were designed and synthesized to perform dPCR amplification. By comparing different primer probes and optimizing reaction conditions, a preliminary RT-ddPCR method capable of detecting NIV was established. Subsequently, the linearity and repeatability of the method were examined, and the method was compared on different platforms, as well as compared with the two-step method, and detection specificity of the established method.was evaluated. Finally, in virto transcription RNA candidate reference materials were quantified using established RT-ddPCR method. The results showed that the established method had good specificity and repeatability, with a correlation coefficient of 0.9999 between RT-ddPCR method and weight method. There was no significant difference in quantitative results obtained on different platforms, and the quantitative result of one-step method was better than the two-step method. In summary, the RT-ddPCR method established in this study has good specificity and good repeatability and can be used for quantification of NIV reference materials, providing support for quantitative detection of NIV.