Abstract: The research aimed to study the effects of different temperature and reaction time on the antigen titer of H5N1 subtype of avian influenza Re-6 strain determined by hemagglutination test. Three temperatures （4, 25 and 37℃） were set to read the titers after reacting 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 and 60 min respectively. The results showed that the the appearance time of result was shortened with the increase of reaction temperature. The results tended to be stable after reacting at 4℃for 25 min and deflocculation started after reacting 50 min. The results tended to be stable after reacting at 25 ℃ for 20 min and deflocculation started after reacting 40 min. The results tended to be stable after reacting at 37 ℃for 15 min and deflocculation started after reacting 30 min.