We aimed to develop a real-time polymerase chain reaction (PCR) detection method for the Reston subtype of the Ebola virus. The NP gene of the Reston subtype of the Ebola virus was selected as the detection object. Sequences of different subtypes of Ebola viruses were aligned using Clustal W software. The most unique and conserved regions of the Reston subtype of the Ebola virus were recruited as candidate sequences for specific primers. Primer Express and Primer Premier 5. 0 software were used to filter the optimal pair of primers for detection. Real-time PCR was carried out using optimized parameters and positive DNA prepared by serial (tenfold) dilution of a recombinant plasmid and by plotting a standard curve. In addition, the reproducibility, accuracy, and specificity of the assay were tested. Results showed that the sensitivity of detection of the Reston subtype of the Ebola virus by real-time PCR could reached 10(2) copies/microL. The linear relationship (R2) reached 0.997, the slope of the standard curve was -0.3101, and amplification efficiency was 110.145%. A sharp and narrow melting peak appeared at 79.94 degrees C for all standards in different dilutions. In conclusion, a fast and sensitive real-time PCR detection system for the Reston subtype of the Ebola virus was developed. This system could be used as a supplementary diagnostic and monitoring approach for basic and clinical studies on the Reston subtype of the Ebola virus. The detection system does not require expensive technology or specialist operators.