Abstract: Preparation and preliminary application of diagnostic gene chip of several avian diseases have been developed in this paper. Recombinant plasmids of target genes has been designed and cloned respectively. The target genes of NDV, IBV, AIV and IBDV were amplified by PCR with recombinant plasmids templates respectively and purified. Target genes were spotted on glass slides with microarray printing system, and the spotted slide was dried at room temperature, hydrated, lighted with UV and washed. The diagnostic gene chip for detecting NDV, IBV, AIV and IBDV were produced successfully. The results of qualification test showed that the diagnostic gene chip are reliable and well for detecting NDV,IBV, AIV and IBDV, and the sensitivity, specificity and repeatability of this new technology is well for detecting. 30 clinical samples were detected by gene chip technology and RT-PCR at the same time, the results illustrated that the detecting rate for NDV, IBV and IBDV are nearly same between the two ways, but the gene chip technology can detect different disease simultaneously.