Abstract: To establish a reverse transcription droplet digital PCR (RT-ddPCR) detection method for Nipah virus (NIV), a recombinant plasmid was constructed using the conserved sequence of the NIV-N gene as the target gene. This plasmid served as a template to produce in vitro transcribed RNA. Five sets of specific primer-probe pairs were designed and synthesized for digital PCR amplification. By comparing different primer-probe sets and optimizing reaction conditions, a preliminary RT-ddPCR method capable of detecting NIV was established. Subsequently, the linearity and repeatability of the method were evaluated, and the method was compared across different platforms as well as with a two-step method to assess detection specificity. Finally, the established method was used to quantify candidate reference materials of in vitro transcribed RNA. The results demonstrated that the established method possesses excellent specificity and repeatability, with a correlation coefficient of 0.9999 between the RT-ddPCR method and the weight-based method. Quantitative results obtained on different platforms showed no systematic errors, and the one-step method yielded superior quantitative results compared to the two-step method. The established RT-ddPCR method exhibits strong specificity and good repeatability, making it suitable for the quantification of NIV reference materials and providing robust support for the quantitative detection of NIV.