Abstract: Abstract: Many studies have shown that resistant starch has health benefits such as preventing colon cancer, reducing the risk of ulcerative colitis, increasing absorption of minerals, inhibiting fat accumulation and hypoglycemic effects. In this study, the cassava starch was used as raw material. The resistant starch was prepared by autoclaving assisted enzyme treatment. To improve the content of resistant starch, the parameters of enzyme treatment that include adding amounts of α-amylase, reaction time of α-amylase, adding amounts of pullulanase and reaction time of pullulanase were optimized with a response surface methodology. The F-value of 57.58 implies the model is significant. The Lack of Fit F-value of 1.66 implies the Lack of Fit is not significant relative to the pure error. The Pred R-Squared of 0.9156 is in reasonable agreement with the Adj R-Squared of 0.9659 and the R-Squared is 0.9829. This model can be used to navigate the design space. The optimized conditions were that when 10% of the starch slurry concentration was autoclaved at 120℃ for 80min, added 1U α-amylase per gram of starch to react 15.75 min at 95℃, debranched with 0.83U pullulanase per gram of starch at 55℃ for 5.86h, then treated with ultrasonic for 2min, and finally retrograded at 4℃ for 24h to improve resistant starch formation, the content of resistant starch increased to 15.48%. Compared with raw starch, the granule appearance of resistant starch changed under scanning electron microscopy. X-ray diffraction showed that the cassava starch had an A type pattern with sharp peaks at 15°, 17°, 18° and 23°. However, the resistant starch had a B type pattern with sharp peaks at 17° and 22°. The crystallinity of resistant starch improved with increasing resistant starch content. The in vitro digestion results indicated that total carbohydrate digestion products were decreased with increasing resistant starch content and that digestion rates were decreased with increasing incubation time. In first three hours, the concentration and activity of α-amylase solution were high and the binding sites between starch molecules and α-amylase were enough, so the total carbohydrate digestion products of all samples were increased sharply in the initial stage. After three hours, the digestion products were increased slightly and the digestion rates slowed down. This was mainly because starch molecules were hydrolyzed into a small molecular chain and the binding sites were decreased.