Starch is an abundant, renewable, and low cost material that has been extensively studied for its role in crystallization. The aim of this study is to develop a convenient and green approach to synthesize starch nanoparticles (StNPs). Short glucan chains were successfully prepared by using pullulanase that could debranch the amylopectin obtained from waxy maize starch. StNPs were prepared via the self-association of short glucan chains, of which the crystallinity structure changed from A-type (native starch) to B-type (starch nanoparticles) through the enzymatic hydrolysis and reassembly process at 4°C. Scanning electron microscopy (SEM), X-ray diffraction (XRD), dynamic light scattering (DLS) and differential scanning calorimetry (DSC) were used to characterize the morphology and crystalline structure of StNPs. The results showed that the diameter of StNPs ranged from 300 nm to 1.5 μm, depending on the initial concentration of short glucan chains and self-assembly time. The developed approach could produce well-defined and uniform starch nanoparticles that could readily be employed to encapsulate various functional guest molecules in biocompatible starch based nanoparticles in food industry.