2-O-α-D-glucopyranosyl L-ascorbic acid(AA-2G) was transformed by marine microorganism cyclodextrin glucanotrans-ferase (CGTase) using L-ascorbic acid and β-cyclodextrin(β-CD) as substrate. The qualitative and quantitative analyses of AA-2G were undertaken by LC-MS and HPLC. On the basis of single factor experiment Plackett-Burman design was carried out to determine three main effective factors (pH, substrate concentration, enzyme concentration). Then Box-Behnken design using three factors and three levels was applied to optimize synthesis conditions of AA-2G. The results indicated that the optimum conditions were pH 9.07, enzyme concentration 90.78 U/g·β-CD, substrate concentration 56.81 g/L, the ratio of substrate 1∶1(volume ratio), reacting for 24 h, at 45 ℃. Under this condition the yield of AA-2G was 10.62 g/L.