In this study, the starches were isolated from three botanical sources (i.e., rice, sweet potato, and lotus seed). The size distributions of starch granules and molecules were determined by asymmetrical flow field-flow fractionation (AF4), and compared with those measured from optical microscopy (OM) and dynamic light scattering (DLS). Furthermore, the starches were characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), and differential scanning calorimetry (DSC). AF4 coupled online with UV-visible, multiangle light scattering (MALS), and differential refractive index (dRI) detectors (AF4-UV-MALS-dRI) was employed for the investigation of the digestion and retrogradation properties of starches. Meanwhile, the relationships between the size of starch at nano- to microscale and its functional properties (i.e., digestibility, retrogradation, and thermal properties) were studied by Pearson correlation analysis. AF4-UV-MALS-dRI was proved to be a rapid and gentle method for the separation and size characterization of starches at both micro- and nano-molecule levels. Moreover, it was demonstrated that AF4-UVMALS-dRI is a useful tool for the monitoring of the digestion and retrogradation properties of starches. The results suggested that the sizes of starch granules and molecules were to some extent correlated with their thermal properties and digestibility, but not with retrogradation property.