Based on one-factor-at-a-time experiments, the main parameters influencing the quantitive detection of natural resistant starch were optimized by orthogonal test, including reaction time, pH value, reaction temperature, and saccharifying enzyme amount. For the remove of digestible starch, pH of mixed enzyme solution and water-incubation time were 6.0 and 12 h, respectively. For determination of resistant starch, the glucoamylase dosage, water-incubation temperature and time were 300 μL, 55 ℃ and 75 min, respectively.