Abstract: In this study, the effects of starch emulsion concentration, autoclave time and autoclave temperature on the preparation of glutinous millet resistant starch (GMRS) were systematically investigated by using the yield of GMRS as the detection index. Then, the physicochemical properties and microstructure of glutinous millet starch (GMS) before and after autoclave treatment were further analyzed and compared. The influence of different ratio of GMRS addition on the texture and GI value of biscuits were studied. The results showed that the optimum conditions for the preparation of GMRS by autoclave method were starch emulsion concentration of 10%, autoclave time of 40 min, and autoclave temperature of 120 ℃. Under these conditions, the yield of GMRS was 30.64%. Further detection indicated that compared with GMS, the light transmittance, solubility and swelling power of GMRS were decreased significantly. Unlike GMS, which had small particle size and relatively smooth surface, the morphology of GMRS prepared by autoclave method was flaky and rough surface with pore like depression. The data of the further experiments, in which GMRS was used to replace part of the flour to make biscuits, showed that with the increasing of the amount of GMRS, the shear force of biscuits increased, and the GI value decreased significantly, the biscuits could be transformed from high GI value food to medium GI value food, meeting the needs of consumers for low GI value food.