Objective To explore the most suitable condition of influenza virus which was culturing on plateau region,and im-prove the effect of the separation of influenza virus.Methods The original specimens were respective inoculated in MDCK cells,by comparing CO2 percent concentration (5%,4.8%,4.6%,4.4%,4.2%),TPCK-pancreatic enzyme dosage (1,2,3, 4,5μg/m and 6,8,10μg/ml),inoculation amount (100,200,300,400,500μl),incubation time (48,72,96,120,144 h),inoc-ulation methods (adsorption,direct inoculated),and culture vessel(cells,6 orifice)that were influenced on influenza virus, the best culture conditions was determined.Hemagglutinatio (HA)method was used to detect the virus titers.Results Through the comparison,the HA titer of virus was in the highest titer with 6 orifice plate culture vessel,4.4% CO2 and 4.6% CO2 ,2μg/ml TPCK-pancreatic enzyme,300μl and 400μl inoculation amount,72h~96h of incubation time.Conclusion The optimal condition of the influenza virus cultured with the MDCK cell has been established in the laboratory on plateau region.