The effect of ultrasound/CaCl2 co-treatment on aggregation structure, thermal stability, rheological, and film properties of high amylose corn starch (HACS) was investigated. The scanning electron microscopy (SEM) images revealed the number of starch fragments and malformed starch granules increased after co-treatment. The differential scanning calorimetry (DSC) results showed the co-treated HACS got a lower gelatinization temperature (92.65 ± 0.495 °C) and enthalpy values (ΔH, 4.14 ± 0.192 J/g). The optical microscope images indicated that lesser Maltase crosses were observed in co-treated HACS. The results of X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR) indicated ultrasound influenced the compactness of amorphous zone and CaCl2 damaged the crystalline region of HACS granules. Additionally, the rheology properties of HACS dispersion demonstrated the apparent viscosity of co-treated dispersion increased as the ultrasound time prolonged. The mechanical strength and structural compactness of HACS films were improved after ultrasound treatment. The mechanism of ultrasound/CaCl2 co-treatment improved the gelatinization and film-forming ability of HACS was that (i) ultrasound wave loosened the HACS granules shell, promoted the treatment of CaCl2 on HACS granules, and (ii) ultrasound wave improved the uniform distribution of HACS dispersion, increased the interaction between CaCl2 and starch chains during the process of film-forming.