Cyclodextrins are widely used for various purposes and limited because of it′s low solubility.Branched cyclodextrins have attracted more and more attention because of their many advantages such as higher solubility in water.In this study a new method for synthesizing maltosyl(α-1→6)β-cyclodextrin with a high yield was described.Mal-β-CD was synthesized through the reverse reaction of Bacillus licheniformis pullulanase and identified.The factors which affect the formation of maltosyl(α1→6)β-cyclodextrin were studied and the optimum conditions were obtained as follows: 250 U/g β-CD of enzyme amount,molar ratio of maltose to β-CD 12∶1~16∶1,a substrate mass fraction of 80%,the pH of reaction mixture 4.5,reaction temperature 70 ℃ and reaction time 60 h respectively.The product was separated by Sephadex G25 column and further purified by Bio-gel P-2 column.Maltosyl(α-1→6)β-cyclodextrin was identified by IR,ESI-MS and NMR.It was proved to be a effective method to synthesize maltosyl(α-1→6)β-cyclodextrin by using Bacillus licheniformis pullulanase.The yield of Mal-β-CD was about 56% under the optimal conditions.