To explore the influence of the distribution of substituents within starch granules on the physicochemical properties of waxy corn starch, waxy corn starch was used as the raw material. Enzymatic hydrolysis was performed using pullulanase and glucoamylase. Enzymatically hydrolyzed acetylated starches with different degrees of modification were prepared by controlling the conditions of enzymatic hydrolysis and acetylation modification. The enzymatically hydrolyzed acetylated starches were subjected to surface gelatinization treatment. The changes in the degree of substitution in different regions within the granules were determined by the degree of gelatinization and gel permeation chromatography. The starches with different substituent distributions were analyzed by scanning electron microscopy, differential scanning calorimetry, infrared spectroscopy, and X-ray diffractometry. The freeze-thaw stability of starches with different substituent distributions was measured. The results showed that based on the analysis of the gelatinization degree of starch granules and the degree of substitution before and after gelatinization, with the increase in the degree of enzymatic hydrolysis and modification, the degree of substitution of the remaining starch after gelatinization increased, acetyl groups were introduced into the interior of starch granules, resulting in a relatively uniform distribution of the substituents in the starch granules. The scanning electron microscopy results indicated that as the degree of modification deepened, the surface structure of starch granules was damaged, with depressions and holes appearing, and the internal substitution of starch granules increased. The results of infrared spectroscopy, differential scanning calorimetry, X-ray diffractometry, and freeze-thaw stability analyses showed that as the degree of acetyl substitution inside the starch granules increased, the glass transition temperature of starch molecules decreased, the characteristic crystallization peaks weakened, the relative crystallinity decreased, and the freeze-thaw stability enhanced. The purpose of this study was to provide a reference for the further application of composite modified starch in the food industry and the subsequent study of substituent distribution.