Abstract: In order to establish a one-step multiplex RT-PCR detection method for rapid identification of PRRSV, genotype 2 CSFV, PRV and JEV, the experiment used PRRSV ORF6 gene, genotype 2 CSFV E2 gene, PRV gE gene and JEV E gene as target genes to construct recombinant nucleic acid standards. Four pairs of specific detection primers were designed, and the One-step RT-PCR Enzyme Mix concentration, primer concentration and annealing temperature in the same reaction system were optimized to test specificity, sensitivity and repeatability. The known clinical samples（35 PRRSV, 45 CSFV, 20 PRV（gE） and 25 JEV positive nucleic acid and 20 negative nucleic acid samples） were detected and the compliance rate was compared. The results showed that the optimal amplification system of one-step multiplex RT-PCR was determined by optimizing the reaction conditions: 5×QIANGEN One-step RT-PCR Buffer 10 μL, dNTP Mix 2 μL, One-step RT-PCR Enzyme Mix（20 U/μL） 2 μL, upstream and downstream primers（genotype 2 CSFV and JEV primer concentration of 5 μmol/L, PRRSV primer concentration of 3 μmol/L, PRV primer concentration of 7 μmol/L） each 1 μL, template 1 μL, DEPC water was made up to 50 μL. Amplification program was 50 ℃ for 30 min, 95 ℃ for 15 min, 94 ℃ for 30 s, 58 ℃ for 30-45 s（genotype 2 CSFV, PRRSV 30 s, PRV, JEV 45 s）, 72 ℃ for 1 min, a total of 35 cycles; 72 ℃ for 10 min. Genotype 1 CSFV strain（Shimen strain）, CSFV vaccine strain（HCLV strain）, PRV vaccine strain, BVDV and 9 common swine viruses and bacterial pathogens were not detected by this method. The detection limits of PRRSV, genotype 2 CSFV, PRV and JEV nucleic acid standards were 1.72×10~2 copies/μL, 1.97×10~3 copies/μL, 1.94×10~3 copies/μL and 2.08×10~2 copies/μL, respectively; the method had good repeatability; the coincidence rates with the detection results of commercial kits were PRRSV 100%（k=1）, genotype 2 CSFV 96.9%（k=0.93）, PRV 100%（k=1） and JEV 100%（k=1）. The results suggested that this study established a rapid, accurate and efficient one-step multiplex RT-PCR detection method that could simultaneously detect PRRSV, genotype 2 CSFV, PRV（gE gene） and JEV, and had good specificity, sensitivity and repeatability.