Abstract: In order to investigate the changes in structure and properties of waxy maize starch (WMS) after its nano-starch was prepared by pullulanase enzymolysis-dynamic high pressure micro-fluidization technology (P-DHPM), this paper explored the trends of changes in starch microstructure, crystalline structure, molecular structure, particle size, molecular weight, and thermal properties before and after the treatment. The results showed that spherical starch particles were turned into fragmentization, and the crystalline structures were transferred into amorphous states, the short-range ordered structure was increased, and the molecular weight of starch was decreased with the increment of enzymatic hydrolysis time. In the meantime, the peak gelatinization temperature (T_p) of treated starch was decreased from 71.23 ℃ to 55.81 ℃, and the enthalpy value of pasting (ΔH) was decreased to 4.68 J/g significantly (P<0.05). Furthermore, with the treatment of DHPM, the particle size of pre-treated starch was significantly (P<0.05) reduced to nano-scale, presenting more random small pieces compared with enzymatic starch. Comparativley, the T_p of nanostarch were reduced to 55.37 °C, while the ΔH of nanostarch was increased to 14.13 J/g further. These results indicated that nano-starch with short-range ordered structure could be prepared from P-DHPM, providing an environmentally friendly and easy-to-operate preparation method, which would provide a foundation for further study of its application in starch nano-emulsion and starch-based active packaging materials.