This study provides a novel method of preparing lotus seed starch nanocrystals (LS-SNCs) using acid hydrolysis combined with ultrasonic-assisted acid hydrolysis (U-LS-SNCs) and evaluates the structural characteristics of starch nanocrystals using scanning electron microscopy; analysis of particle size, molecular weight, and X-ray diffraction patterns; and FT-IR spectroscopy. The results showed that the preparation time of U-LS-SNCs could be reduced to 2 days less than that for LS-SNCs. The smallest particle size and molecular weight were obtained after a 30 min treatment with 200 W of ultrasonic power and 5 days of acid hydrolysis. The particle size was 147 nm, the weight-average molecular weight was 3.42 × 104Da, and the number-average molecular weight was 1.59 × 104Da. When the applied ultrasonic power was 150 W for 30 min and acid hydrolysis was applied for 3 days, the highest relative crystallinity of the starch nanocrystals was 52.8%. The modified nanocrystals can be more widely used in various applications such as food-packaging materials, fillers, pharmaceuticals, etc.