A cold-adapted (ca), temperature sensitive (ts), live attenuated influenza B virus strain B/Ann Arbor/1/66 was chosen for influenza virus rescue research, in which six internal gene segments, PB1, PB2, PA, NP, M, NS, were fully synthesized and nine amino acid substitutions were artificially alter by human intervention. The resultant B/Ann Arbor/1/66 plasmids were named as pAB121-PB1, pAB122-PB2, pAB123-PA, pAB124-HA, pAB125-NP, pAB126-NA, pAB127-M and pAB128-NS, respectively. A recombinant influenza A virus was previously generated entirely from cloned cDNA. An infectious recombinant influenza B virus was generated here, and designated as rMDV-B, by plasmid-based reverse genetics. The rMDV-B virus contained HA and NA genes from an epidemic influenza B virus strain B/Malaysia/2506/2004 in the background of internal genes derived from influenza B virus strain B/Ann Arbor/1/66. HA titer of rMDV-B in MDCK cells and embryonated chicken eggs ranged from 1∶64 to 1∶512. The results may allow an effective live influenza B vaccine to be produced from a single master strain, providing a model for the design of future live human influenza vaccines.