¹College of Animal Science and Veterinary Medicine, Henan Institute of Science and Technology, Xinxiang 453003, CHINA DOI : 10.9775/kvfd.2020.24110 In the present study, a duplex SYBR Green real-time PCR assay was developed in order to indentify Actinobacillus pleuropneumoniae and Haemophilus parasuis infection in one reaction, through a melting curve analysis. This method utilized two pairs of specific primers that allowed the amplification of highly conserved regions of A. pleuropneumoniae Apx IV and H. parasuis omp P2 gene. Reconstitution experiments were conducted by using PMD - 19T plasmid in order to determine the sensitivity of the assay. The results showed that the Tm values of the melting curves of A. pleuropneumoniae and H. parasuis were 83.36±0.09ºC and 76.48±0.17ºC, respectively that could accurately distinguish these two pathogens. And no cross reaction were observed between other respiratory pathogens, which suggested a high specificity of two primers. The detection sensitivity of the assay was 127 and 96 copies/μL which was higher than that of the ordinary PCR detection methods. This rapid technique may present a simple, useful option for simultaneous detection of A. pleuropneumoniae and H. parasuis, which would be feasible and attractive for clinical samples diagnosis and epidemiological investigations. Keywords : Actinobacillus pleuropneumoniae, Haemophilus parasuis, SYBR Green Ι, Real-time PCR, Melting curve