Abstract: The study aimed to establish a DNA microarray to simultaneously detecting Actinobacillus pleuropneumoniae, Mycoplasma hyopneumoniae and Pasteurella multocida. 7 different specific target DNA fragments designed and amplified from the genomes of the bacteria were used to construct the DNA microarray. The concentrations of target DNA and probes, hybridization temperature, reproducibility, specificity and sensitivity of the microarray were tested. The microarray could be used repeatedly and possessed high specificity. Microarray hybridization patterns of A. pleuropneumoniae, M. hyopneumoniae and P. multocida could be distinguished from other 9 pathogens species, such as A. lignieresii, Haemophilus parasuis, M. hyorhinitis. The sensitivity of the microarray was determined to 1050 pg of genome DNA in 50 μL labeling reaction system. 44 different serotype reference, isolates and vaccine strains of A. pleuropneumoniae, M. hyopneumoniae and P. multocida were detected with the constructed microarray. Their signal intensities and signal to noise ratio (SNRs) were greater than or equal to 1 000 and 6, respectively. Selective cultures of samples from disease pigs (n=45) and clinical healthy pigs (n=97) were detected by the microarray. Recall ratios of P. multocida, A. pleuropneumoniae, M. hyopneumoniae from disease and healthy pigs were 71.1% and 49.5%, 42.2% and 26.8%, 20% and 227%, respectively. Mixed infection rates were 42.2% and 24.7% in disease and healthy pigs respectively. The coincidence ratios between the microarray and PCR were 97.8%100%, and the ratios between the microarray and isolation method were 87.6%95.6%, in detecting clinical samples. Results indicated that the microarray have high specificity and sensitivity, and is a new tool for detection and identification of A. pleuropneumoniae, M. hyopneumoniae and P. multocida.