Plackett-Burman design was used to select reaction conditions for large ring cyclodextrin(LR-CD) preparation,and the significant factors were optimized by Box-Behnken design.Results showed that,enzyme,temperature and time were three significant factors,t-test showed that model of Box-Behnken design was significant(P≤0.05),and lack of fit was no significant(P≥0.05).The regression coefficient R2 was 0.9404,which was showed a good regression result.The predicted yield of LR-CD was 56.58%,and the predicted conditions were enzyme 91.34 U/g,temperature 71.7°C for 6.8 h.Validation test showed LR-CD yield of 55%,2.3 folds higher than initial yield.Identification results showed that the degree of polymerization was ranged from 20 to 40,the ratio of cyclic component was 47%.