H3N2 subtype influenza A viruses have been identified in humans worldwide, raising concerns about their pandemic potential and prompting the development of candidate vaccines to protect humans against this subtype of influenza A virus. The aim of this study was to establish a system for rescuing of a cold-adapted high-yielding H3N2 subtype human influenza virus by reverse genetics.In order to generate better and safer vaccine candidate viruses, a cold-adapted high yielding reassortant H3N2 influenza A virus was genetically constructed by reverse genetics and was designated as rgAA-H3N2. The rgAA-H3N2 virus contained HA and NA genes from an epidemic strain A/Wisconsin/67/2005 (H3N2) in a background of internal genes derived from the master donor viruses (MDV), cold-adapted (ca), temperature sensitive (ts), live attenuated influenza virus strain A/Ann Arbor/6/60 (MDV-A).In this presentation, the virus HA titer of rgAA-H3N2 in the allantoic fluid from infected embryonated eggs was as high as 1:1024. A fluorescent focus assay (FFU) was performed 24-36 hours post-infection using a specific antibody and bright staining was used for determining the virus titer. The allantoic fluid containing the recovered influenza virus was analyzed in a hemagglutination inhibition (HI) test and the specific inhibition was found.The results mentioned above demonstrated that cold-adapted, attenuated reassortant H3N2 subtype influenza A virus was successfully generated, which laid a good foundation for the further related research.