Abstract: In order to optimize the method of determining tobacco pectin using enzymolysis-flow method, reaction temperature, enzymatic hydrolysis time and buffer pH were studied through the Box-Benhnken experimental design and response surface methodology. The results showed that reaction temperature had the greatest impact on the determination of pectin, followed by buffer pH, and the enzymatic hydrolysis time comes last. The optimal enzymatic hydrolysis conditions were pH 3.8, time 1.9 h and 56 ℃. Under this condition, the linear relationship of pectin was optimal (R²=0.999), with the recovery of pectin in tobacco ranged from 99.2% to 100.9%, and the RSD(n=6) was less than 3%. This suggested that the optimized enzymatic hydrolysis conditions used by the Box-behnken design and response surface analysis have good precision and accuracy and are suitable for testing large number of samples.