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As rapid and reliable tools for diagnosis of porcine respiratory disease complex (PRDC) bacterial pathogens are getting more important over time, we established a new multiplex fluorescence quantitative polymerase chain reaction (mFQ-PCR) system with a similar symptom. Our results showed that the system could accurately and simultaneously detect 8 pathogens within 1.5 h. The detection limits were 2-5 standard DNA copies. The correlation coefficient of the standard curve was more than 0.998, with a well dynamic range from 101 to 109, and the intra- and intervariation coefficients (CVs) were less than 2.06%. After setting the positive threshold of Ct value, the coincidence rate between mFQ-PCR and conventional PCR (cPCR) could reach 92%-100%, the specificity and sensitivity of mFQ-PCR exceeded 93.5% and 90.8%, respectively. We also found that the positive rates of 8 bacteria were 12.5%-68.2%, while the rates of co-infection were 90%-100% in the clinical samples. The co-infection among Streptococcus suis (Ss), Haemophilus parasuis (Hps), and Mycoplasma hyorhinis (Mhr) accounted for 78.3%, 91.4%, 100%, respectively of all the positive samples, among Actinobacillus pleuropneumoniae (App) and Mycoplasma pneumoniae (Mhp) are 45.5% and 35.7%, among Bordetella bronchis (Bb), Pasteurella multocida (Pm) and toxic Pasteurella multocida (T+Pm) are 52.2%, 41.4%, and 100%, respectively. The results demonstrated that co-infection of 2-4 pathogenic bacteria with similar symptoms was a common situation for PRDC in south of China; our method has the potential to become a reliable detection tool. [ABSTRACT FROM AUTHOR]