Abstract: In order to diagnose quickly tiger influenza in clinic, using the tiger influenza virus (TIV) sequence and the influenza A virus sequence information available in the Genbank data base, the primers were selected from conserved region of a target gene specific for TIV type (NP) and subtype (HA,NA) for use in RT-PCR. Specific primers were desiged for NP, HA, NA gene with Primer5.0 primer design software, DNA fragments of sizes 464, 581 and 173 bp, respectively, were amplified. The primers to be in common used for influenza A virus were desiged for use in the synthesis of cDNAs. Optimize the reaction condition and establish a multiplex RT-PCR in a single step for simultaneous detection of the TIV 3 genes fragments. The assay were applied in clinical diagnosis. This decision was made following the results of a comparison with electron microscope, hemagglutination /hemagglutination inhibition and isolation of the virus in tissue culture or allantoic fluid. The results were that the sensitivity of detection was about 100 TCID50, an excellent concordance between diagnosis by the RT-PCR and culture was demonstrated, and the RT-PCR in detection of TIV is more sensitive and rapid than above mentioned methods. So the method described here is very practicable and valuable in clinical diagnosis or in laboratory application.