【Objective】A method of detecting avian influenza virus（AIV） and separating H1 and H3 subtype was determined to provide references for effectively preventing and controlling AI. 【Method】Three sets of specific primers were designed according to the conserved sequences of AIV matix （M） gene， and the hemagglutinin （HA） genes of H1 and H3 subtype AIV. A multiplex PCR assay system was developed and optimized for rapid and simultaneous detection of these genes. The specificity and sensitivity of this method was evaluated. This method was used to test the AIV infection from clinical samples. 【Result】It was shown that all samples containing H1 and H3 subtype AIV could be amplified into three specific bands by this multiplex PCR. The lengths of these bands were 302 bp H1 AIV HA，453 bp（M） and 626 bp（H3 AIV），respectively. Samples containing H1 or H3 subtype AIV could be amplified into two specific bands， which were 302 bp and 453 bp as well as 453 bp and 626 bp， respectively. Samples containing other subtypes AIV could be amplified into a 453 bp specific band. However， no specific band was amplified from other avian pathogenic virus. As little as 7.00 pg of H1 subtype AIV， 3.62 pg of M subtype and 20.60 pg of H3 subtype AIV could be detected by this multiplex PCR. Thirty-six clinical samples （36/100， 36%） were found to be infected with AIV， of which 4 were H1 subtype AIV， 9 were H3 subtype AIV and 23 were other type AIV. 【Conclusion】Detecting H1， H3 and M subtypes of avian influenza by multiplex PCR was appropriate， rapid， sensitive and specific.